Methods of determining a surgical margin and methods of use thereof

ABSTRACT

Provided herein are methods of determining a surgical margin and the site and size of a tissue to be resected from a subject, and methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/549,175, filed Dec. 13, 2021, which is a continuation ofInternational Application PCT/US2021/036415, with an internationalfiling date of Jun. 8, 2021 which claims priority to U.S. ProvisionalPatent Application No. 63/036,195, filed Jun. 8, 2020. The entirecontents of the foregoing application are incorporated herein byreference.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

SUMMARY

Provided herein are method of identifying a surgical margin of a tissueto be resected in a subject, the method comprising: (a) contacting atissue sample obtained from the subject to an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises (i) a capture domain that specifically binds toan analyte of the tissue sample and (ii) a spatial barcode; (b)determining (i) all or a part of a nucleic acid sequence correspondingto the analyte specifically bound to the capture domain or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (c) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin of the tissue to be resected from the subject basedon the comparison.

Also provided herein are methods of determining size and site of atissue to be resected from a subject, the method comprising: (a)contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises (i) a capture domain that bindsspecifically to an analyte of the tissue sample and (ii) a spatialbarcode; (b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (c)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at a different location in the tissuesample, and determining the size and site of the tissue to be resectedfrom the subject based on the comparison.

Also provided herein are methods of reducing the risk of re-excision ofa tissue, the method comprising: (a) contacting a tissue sample to anarray comprising a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode; (b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (c)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Also provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject, the method comprising: resecting tissuefrom the subject using a surgical margin previously determined using amethod comprising the steps of: (a) contacting a tissue sample obtainedfrom the subject to an array comprising a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises a(i) capture domain that specifically binds to an analyte of the tissuesample and (ii) a spatial barcode; (b) determining (i) all or a part ofa nucleic acid sequence corresponding to the analyte specifically boundto the capture domain or a complement thereof, and (ii) all or a part ofa nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (c) comparing the presence of the analyte at thelocation in the tissue sample to presence of the analyte at differentlocation(s) in the tissue sample, and determining the surgical marginbased on the comparison.

In some embodiments, the array comprises a slide. In some embodiments,the array is a bead array.

In some embodiments, step (b) comprises sequencing (i) all or a part ofthe nucleic acid sequence corresponding to the analyte specificallybound to the capture domain or a complement thereof, and (ii) all or apart of the nucleic acid sequence corresponding to the spatial barcodeor a complement thereof. In some embodiments, the sequencing is highthroughput sequencing. In some embodiments, step (b) comprises extendinga 3′ end of the capture probe using the specifically bound analyte as atemplate to generate an extended capture probe. In some embodiments,step (b) further comprises generating a single-stranded nucleic acidcomprising a nucleic acid sequence that is complementary to all or apart of the extended capture probe.

Also provided herein are methods of identifying a surgical margin of atissue to be resected, the method comprising: (a) contacting a tissuesample to a plurality of analyte capture agents, wherein an analytecapture agent of the plurality of analyte capture agents comprises ananalyte binding moiety barcode, an analyte capture sequence, and ananalyte binding moiety that binds specifically to an analyte; (b)disposing the tissue sample onto an array, wherein the array comprises aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain thatbinds specifically to the analyte capture sequence; (c) determining (i)all or a part of a nucleic acid sequence corresponding to the analytebinding moiety barcode or a complement thereof, and (ii) all or a partof a nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (d) comparing the presence of the analyte at thelocation in the tissue sample to presence of the analyte at differentlocation(s) in the tissue sample, and determining the surgical margin ofthe tissue to be resected based on the comparison.

Also provided herein are methods of determining size and site of atissue to be resected from a subject, the method comprising: (a)contacting a tissue sample obtained from the subject to a plurality ofanalyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte; (b) disposing the tissue sample ontoan array, wherein the array comprises a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain that binds specifically to theanalyte capture sequence; (c) determining (i) all or a part of a nucleicacid sequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing the presence of the analyte at the location in the tissuesample to the presence of the analyte at different location(s) in thetissue sample, and determining the size and site of the tissue to beresected from the subject based on the comparison.

Also provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject, the method comprising: resecting tissuefrom the subject using a surgical margin previously determined using amethod comprising the steps of: (a) contacting a tissue sample obtainedfrom the subject to a plurality of analyte capture agents, wherein ananalyte capture agent of the plurality of analyte capture agentscomprises an analyte binding moiety barcode, an analyte capturesequence, and an analyte binding moiety that binds specifically to ananalyte; (b) disposing the tissue sample onto an array, wherein thearray comprises a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises a spatial barcode and acapture domain that binds specifically to the analyte capture sequence;(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (d) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin based on the comparison.

In some embodiments, step (c) comprises sequencing (i) all or a part ofthe nucleic acid sequence corresponding to the analyte binding moietybarcode or a complement thereof, and (ii) all or a part of the nucleicacid sequence corresponding to the spatial barcode or a complementthereof. In some embodiments, the sequencing is high throughputsequencing. In some embodiments, step (c) comprises extending a 3′ endof the capture probe using the specifically bound analyte capture agentas a template to generate an extended capture probe. In someembodiments, step (c) further comprises generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe.

In some embodiments, the array comprises a slide. In some embodiments,the array comprises a slide having the plurality of capture probes. Insome embodiments, the array is a bead array.

In some embodiments, the tissue to be resected is a tumor. In someembodiments, the tissue to be resected is infected tissue, necrotictissue, or diseased tissue. In some embodiments, the resected tissue isa tumor. In some embodiments, the resected tissue is infected tissue,necrotic tissue, or diseased tissue.

In some embodiments, the analyte is RNA. In some embodiments, the RNA ismRNA. In some embodiments, the analyte is DNA. In some embodiments, theDNA is genomic DNA.

In some embodiments, the subject is suspected of or diagnosed as havinga cancer. In some embodiments, the cancer is breast cancer. In someembodiments, the subject is suspected of or diagnosed as having ductalcarcinoma in situ. In some embodiments, the analyte is a protein. Insome embodiments, the protein is an intracellular protein. In someembodiments, the protein is an extracellular protein. In someembodiments, the analyte binding moiety is an antibody or anantigen-binding antibody fragment.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 4 is a schematic showing an exemplary method of determining asurgical margin (e.g., a tumor margin) of a tissue to be resected from asubject.

DETAILED DESCRIPTION

This application is based on the discovery of a method of analyzingspatial expression profiles of analytes in tissue sections and itsapplications on determining surgical margins and methods of treatingpatients in need thereof.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence complementary to a sequence of anucleic acid analyte, a portion of a connected probe described herein, acapture handle sequence described herein, and/or a methylated adaptordescribed herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 201 contains acleavage domain 202, a cell penetrating peptide 203, a reporter molecule204, and a disulfide bond (—S—S—). 205 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.Cleavable capture probe are further described in WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663, each of which isincorporated by reference in its entirety.

For multiple capture probes that are attached to a common array feature,the one or more spatial barcode sequences of the multiple capture probescan include sequences that are the same for all capture probes coupledto the feature, and/or sequences that are different across all captureprobes coupled to the feature.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 3, the feature 301 can be coupled tospatially-barcoded capture probes, wherein the spatially-barcoded probesof a particular feature can possess the same spatial barcode, but havedifferent capture domains designed to associate the spatial barcode ofthe feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 302. One type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a poly(T)capture domain 303, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 302 in combination with a random N-mer capture domain 304 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 302 in combination with a capture domaincomplementary to a capture handle sequence of an analyte capture agentof interest 305. A fourth type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a capturedomain that can specifically bind a nucleic acid molecule 306 that canfunction in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 3,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 3 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MEW multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents. See, e.g.,Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663.

Capture probes attached to a single array feature can include identical(or common) spatial barcode sequences, different spatial barcodesequences, or a combination of both. Capture probes attached to afeature can include multiple sets of capture probes. Capture probes of agiven set can include identical spatial barcode sequences. The identicalspatial barcode sequences can be different from spatial barcodesequences of capture probes of another set.

The plurality of capture probes can include spatial barcode sequences(e.g., nucleic acid barcode sequences) that are associated with specificlocations on a spatial array. For example, a first plurality of captureprobes can be associated with a first region, based on a spatial barcodesequence common to the capture probes within the first region, and asecond plurality of capture probes can be associated with a secondregion, based on a spatial barcode sequence common to the capture probeswithin the second region. The second region may or may not be associatedwith the first region. Additional pluralities of capture probes can beassociated with spatial barcode sequences common to the capture probeswithin other regions. In some embodiments, the spatial barcode sequencescan be the same across a plurality of capture probe molecules.

In some embodiments, multiple different spatial barcodes areincorporated into a single arrayed capture probe. For example, a mixedbut known set of spatial barcode sequences can provide a strongeraddress or attribution of the spatial barcodes to a given spot orlocation, by providing duplicate or independent confirmation of theidentity of the location. In some embodiments, the multiple spatialbarcodes represent increasing specificity of the location of theparticular array point.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section(II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationsproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

II. Spatial Analysis

Cancer and disease diagnosis and treatment plans are oftentimes guidedby three diagnostic tools: blood work, imaging, and/or biopsies.Oftentimes, surgery is the first line of treatment when cancerous anddiseased tissues are identified, especially in the early stages of thedisease. When surgery is indicated, clinicians face difficult decisionson the size and site of the resection of the diseased tissue. Conclusivediagnosis and staging are oftentimes obtained after surgical resectionhas been completed. Unfortunately, sometimes the entire abnormal tissueis not completely removed, leaving tissue margins that contain diseasedcells that can proliferate in the body and cause continued cancer anddisease.

The methods described here can help support a clinician's decision onthe type of surgical intervention to provide a subject with a potentialcancer or diseased tissue. For example, information from practicing thedescribed methods can help determine the size and site of tissueresection by more completely identifying abnormal tissue margins,thereby reducing the risk of re-excision and/or reducing the risk offuture recurrence of the cancerous or diseased tissue in the subject.

(a) Methods of Determining the Size and Site of a Tissue to be Resected

Provided herein are methods of determining the size and site of a tissueto be resected from a subject that include: (a) contacting a tissuesample obtained from the subject to an array comprising a plurality ofcapture probes, where a capture probe of the plurality of capture probescomprises (i) a capture domain that binds specifically to an analyte ofthe tissue sample and (ii) a spatial barcode; (b) determining (i) all ora part of a nucleic acid sequence corresponding to the analytespecifically bound to the capture domain or a complement thereof, and(ii) all or a part of a nucleic acid sequence corresponding to thespatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (c) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe size and site of a tissue to be resected from the subject based onthe comparison. In some embodiments, the analyte is a DNA or RNA. Insome embodiments, the analyte is a messenger RNA (mRNA) molecule. Insome embodiments, the analyte is a genomic DNA. In some embodiments, theanalyte comprises a full-length sequence of a biomarker describedherein. In some embodiments, the analyte comprises a fragment of thesequence of a biomarker described herein. In some embodiments of any ofthe methods described herein, each of the plurality of capture probescomprises (i) a capture domain that binds specifically to an analyte ofthe tissue section and (ii) a spatial barcode. The capture probe can beany capture probe described herein. In some embodiments, the capturedomain of the capture probe comprises a sequence that is substantiallycomplementary (e.g., at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or 100% complementary) to a portion of the sequenceof the analyte of the tissue sample. In some embodiments, the capturedomain can have a total of about 10 nucleotides to about 125 nucleotides(or any of the subranges of this range described herein). In someembodiments, the sequence that is substantially complementary to aportion of the sequence of the analyte can be a random sequence. In someembodiments, the sequence that is substantially complementary to aportion of the sequence of the analyte can include a poly(T)oligonucleotide sequence (e.g., at least 5 contiguous Ts, at least 10contiguous Ts, or at least 15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by synthesis, sequencing by hybridization, sequencing byligation or any of the other methods for sequencing described herein orknown in the art. For example, sequencing can involve one or more ofnucleic acid amplification, the ligation or addition of one or moresequencing adaptors, cleavage of the capture probe from the array,extension of the capture probe using the bound cDNA as a template, andgenerating a single-stranded nucleic acid comprising a sequence that iscomplementary to all or a part of the extended capture probe.Non-limiting methods for determining the sequence of (i) all or a partof the sequence of the target nucleic acid, or a complement thereof, or(ii) all or a part of the nucleic acid sequence corresponding to thebinding moiety barcode, or a complement thereof, are described herein orare known in the art.

Also provided herein are methods of determining the size and site of atissue to be resected from a subject that include: (a) contacting atissue sample obtained from the subject to a plurality of analytecapture agents, where an analyte capture agent of the plurality ofanalyte capture agents comprises an analyte binding moiety barcode, ananalyte capture sequence, and an analyte binding moiety that bindsspecifically to an analyte; (b) disposing the tissue sample onto anarray, where the array comprises a plurality of capture probes, where acapture probe of the plurality of capture probes comprises a spatialbarcode and a capture domain that binds specifically to the analytecapture sequence; (c) determining (i) all or a part of the nucleic acidsequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the binding moiety barcode or a complement thereof, andusing the determined nucleic acid sequences of (i) and (ii) to identifya presence of the analyte at a location in the tissue sample; (d)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the size and site of a tissue to be resectedfrom the subject based on the comparison. In some embodiments, theanalyte is a protein. In some embodiments, the analyte is a full-lengthprotein. In some embodiments, the analyte is a fragment of a protein. Insome embodiments, the analyte is a byproduct of a protein. In someembodiments, the protein is any of the exemplary cancer biomarkersdescribed herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundanalyte capture sequence as a template, and generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe. Non-limiting methods for determiningthe sequence of (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode, or a complementthereof, or (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode, or a complement thereof, aredescribed herein or are known in the art.

In some embodiments, the tissue to be resected is a tumor (e.g., amalignant or a benign tumor). In some embodiments, the tumor is a solidtumor. In some embodiments, the subject is suspected of having a cancer.In some embodiments, the subject has been previously diagnosed oridentified as having a cancer (e.g., any of the exemplary cancersdescribed herein).

Non-limiting examples of cancers referred to in any one the methodsdescribed herein include: sarcomas, carcinomas, adrenocorticalcarcinoma, AIDS-related cancers, anal cancer, appendix cancer,astrocytomas, atypical teratoid/rhabdoid tumor, basal cell carcinoma,bladder cancer, brain stem glioma, brain tumors (including brain stemglioma, central nervous system atypical teratoid/rhabdoid tumor, centralnervous system embryonal tumors, astrocytomas, craniopharyngioma,ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma,pineal parenchymal tumors of intermediate differentiation,supratentorial primitive neuroectodermal tumors, and pineoblastoma),breast cancer, bronchial tumors, cancer of unknown primary site,carcinoid tumor, carcinoma of unknown primary site, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, cervical cancer, childhood cancers, chordoma, coloncancer, colorectal cancer, craniopharyngioma, endocrine pancreas isletcell tumors, endometrial cancer, ependymoblastoma, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranialgerm cell tumor, extragonadal germ cell tumor, extrahepatic bile ductcancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinalstromal tumor (GIST), gestational trophoblastic tumor, glioma, head andneck cancer, heart cancer, hypopharyngeal cancer, intraocular melanoma,islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cellhistiocytosis, laryngeal cancer, lip cancer, liver cancer, lung cancer,malignant fibrous histiocytoma bone cancer, medulloblastoma,medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skincarcinoma, mesothelioma, metastatic squamous neck cancer with occultprimary, mouth cancer, multiple endocrine neoplasia syndromes, multiplemyeloma, multiple myeloma/plasma cell neoplasm, mycosis fungoides,myelodysplastic syndromes, myeloproliferative neoplasms, nasal cavitycancer, nasopharyngeal cancer, neuroblastoma, non-melanoma skin cancer,non-small cell lung cancer, oral cancer, oral cavity cancer,oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors,ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor,ovarian low malignant potential tumor, pancreatic cancer,papillomatosis, paranasal sinus cancer, parathyroid cancer, pelviccancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors ofintermediate differentiation, pineoblastoma, pituitary tumor,pleuropulmonary blastoma, primary hepatocellular liver cancer, prostatecancer, rectal cancer, renal cancer, renal cell (kidney) cancer, renalcell cancer, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, Sezary syndrome, small cell lung cancer, smallintestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamousneck cancer, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymiccarcinoma, thymoma, thyroid cancer, transitional cell cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor, ureter cancer, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilm'stumor.

In some embodiments, the tissue to be resected can include a tumor(e.g., a malignant tumor) of any of the types of cancer describedherein.

In some embodiments of any one of the methods described herein, theanalyte is a tumor biomarker. In some embodiments, the analyte is atumor antigen. Exemplary tumor antigens include, but are not limited to,melanoma-associated antigen (MAGE) series of antigens (e.g., MAGE-C1(cancer/testis antigen CT7), MAGE-B1 antigen (MAGE-XP antigen, DAM10),MAGE-B2 antigen (DAME), MAGE-2 antigen, MAGE-4a antigen, and MAGE-4bantigen), tyrosinase, glycoprotein 100 (gp100), disialoganglioside GD-2,disialoganglioside O-acetylated GD-3, ganglioside GM-2, epidermal growthfactor receptor (EGFR), vascular endothelial growth factor receptor(VEGFR), mutant B-Raf antigen associated with melanoma and colon cancer,human epidermal growth factor receptor-2 (HER-2/neu) antigen,melanoma-associated antigen recognized by T cells (MART-1) (e.g., MART-126-35 peptide or MART-1 27-35 peptide), protein kinase C-bindingprotein, reverse transcriptase protein, A-kinase-anchoring protein (AKAPprotein), vaccinia-related kinase Serine/Threonine Kinase 1(VRK1),fucosyltransferase (T6-7), zinc finger protein 258 (T11-6), p53-bindingprotein (T1-52), T5-15 (KIAA1735), T5-13 (Sos1), T11-5 (hypotheticalprotein MGC4170), T11-9 (hypothetical protein AF225417), T11-3 (trapankyrin repeat), T7-1 (KIAA1288), a mutant or wild type ras peptide,Homo sapiens telomerase ferment (hTRT), cytokeratin-19 (CYFRA21-1),squamous cell carcinoma antigen 1 (SCCA-1), protein T4-A, squamous cellcarcinoma antigen 2 (SCCA-2), ovarian carcinoma antigen CA125 (1A1-3B)(KIAA0049), cell surface-associated MUCIN 1 (e.g., tumor-associatedMUCIN, carcinoma-associated MUCIN, polymorphic epithelial MUCINpeanut-reactive urinary MUCIN, polymorphic epithelial mucin (PEM), PEMT,episialin, tumor-associated epithelial membrane antigen, epithelialmembrane antigen (EMA), H23 antigen (H23AG), PUM, and breastcarcinoma-associated antigen DF3), CTCL tumor antigen sel-1, CTCL tumorantigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9,CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigense57-1, CTCL tumor antigen se89-1, prostate-specific membrane antigen,5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi'ssarcoma-associated herpesvirus, colon cancer antigen NY-CO-45, lungcancer antigen NY-LU-12 variant A, cancer associated surface antigen,adenocarcinoma antigen ART1, paraneoplastic associatedbrain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplasticneuronal antigen), neuro-oncological ventral antigen 2 (NOVA2),hepatocellular carcinoma antigen gene 520, tumor-associated antigenCO-029, tumor-associated antigen MAGE-X2, synovial sarcoma antigen, Xbreakpoint 2, squamous cell carcinoma antigen recognized by T cell,serologically defined colon cancer antigen 1, serologically definedbreast cancer antigen NY-BR-15, serologically defined breast cancerantigen NY-BR-16, chromogranin A, parathyroid secretory protein 1,pancreatic cancer-associated antigen (DUPAN-2), carbohydrate antigen CA19-9, carbohydrate antigen CA 72-4, carbohydrate antigen CA 195, andcarcinoembryonic antigen (CEA).

In some embodiments, the tissue to be resected is an infected tissue, anecrotic tissue, or a diseased tissue. In some embodiments, the analytecan be associated with an infection, necrosis, inflammation, or disease.Non-limiting examples of such analytes are known in the art.

In some embodiments, the tissue to be resected is infected by abacterium. In some embodiments, the tissue to be resected is infected bya virus. In some embodiments, the tissue to be resected is infected by afungus. In some embodiments, the tissue to be resected is infected by aparasite or protozoa.

In some embodiments, the tissue to be resected is infected by abacterium, e.g., a Bordetella pertussis, a Brucella abortis, aEscherichia coli, a Salmonella species, e.g., a Salmonella typhi, aStreptococci, a Vibrio (V. cholera, V. parahaemolyticus), a Shigella, aPseudomonas, a Brucella species, a Klebsiella, a Mycobacteria species (atuberculosis, an avium, a BCG, a leprosy), a Pneumococci, aStaphylococci, a Enterobacter species, a Clostridium tetani, a Bacillusanthracia, a Streptococcus pneumoniae, a Meningococcus A, B, C, Y, W, orW-135, a Helicobacter pylori, a Rochalimaea henselae, a Pasteurella (P.haemolytica, P. multocida), a Chlamydia (C. trachomatis, C. psittaci), aTreponema pallidum, a Haemophilus species, e.g., a Haemophilus influenzatype b, a mycoplasma species, a Borrelia burgdorferi, a Legionellapneumophila, a Clostridium botulinum, a Corynebacterium diptheriae, aYersinia entercolitica, a Ehrlichia, a Anaplasma, or a Coxiella burnetiibacterium.

In some embodiments, the tissue to be resected is infected by a parasiteor protozoa, e.g., those causing malaria (Plasmodium falciparum, P.vivax, or P. malariae), a schistosome, a trypanosome, leishmania, afilarial nematode, Trichomonas vaginalis, a sarcocystis, a Taeniaspecies (T .saginata or T. solium), Toxoplasma gondi, Trichinellaspiralis, or an Eimeria species.

In some embodiments, the tissue to be resected is infected by a fungus,e.g., Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus,Coccidioides immitis, or Coccidioides posadasii.

In some embodiments, the tissue to be resected is infected by a virus,e.g., a rotavirus, an aphthovirus (the agent for foot and mouthdisease), an Ebola virus, a Hanta virus, a parainfluenza, a herpes virusspecies (e.g., herpes simplex virus, Epstein-Barr virus, chicken poxvirus, pseudorabies, or cytomegalovirus), a rabies virus, a polio virus,a Hepatitis A, B, C or E, distemper, a Venezuelan equineencephalomyelitis virus, a feline leukemia virus, a reovirus, arespiratory syncytial virus, a Lassa fever virus, a polyoma virus, acanine parvovirus, a papilloma virus, a flavivirus, a tick borneencephalitis virus, a paramyxovirus (the agent for Rinderpest), arhinovirus, an enterovirus, a Mengo virus, a paramyxovirus (mumps,measles, or respiratory syncytial virus), an avian infectious bronchitisvirus, HTLV 1, HIV-1 or -2, or influenza virus A, B, or C, a lymphocyticchoriomeningitis virus, a parvovirus, an adenovirus, a togavirus, abovine respiratory syncytial virus, a coronavirus, or a JapaneseEncephalitis virus.

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to presenceof the analyte at different location(s) in the tissue sample, anddetermining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., about 0.1-foldto about 90-fold, about 0.1-fold to about 80-fold, about 0.1-fold toabout 70-fold, about 0.1-fold to about 60-fold, about 0.1-fold to about50-fold, about 0.1-fold to about 40-fold, about 0.1-fold to about30-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about15-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about8-fold, about 0.1-fold to about 6-fold, about 0.1-fold to about 5-fold,about 0.1-fold to about 4-fold, about 0.1-fold to about 3-fold, about0.1-fold to about 2-fold, about 0.1-fold to about 1.5-fold, about0.1-fold to about 1-fold, about 0.1-fold to about 0.8-fold, about0.1-fold to about 0.6-fold, about 0.1-fold to about 0.4-fold, about0.1-fold to about 0.2-fold, about 1-fold to about 100-fold, about 1-foldto about 90-fold, about 1-fold to about 80-fold, about 1-fold to about70-fold, about 1-fold to about 60-fold, about 1-fold to about 50-fold,about 1-fold to about 40-fold, about 1-fold to about 30-fold, about1-fold to about 20-fold, about 1-fold to about 15-fold, about 1-fold toabout 10-fold, about 1-fold to about 8-fold, about 1-fold to about6-fold, about 1-fold to about 5-fold, about 1-fold to about 4-fold,about 1-fold to about 3-fold, about 1-fold to about 2-fold, about 1-foldto about 1.5-fold, about 5-fold to about 100-fold, about 5-fold to about90-fold, about 5-fold to about 80-fold, about 5-fold to about 70-fold,about 5-fold to about 60-fold, about 5-fold to about 50-fold, about5-fold to about 40-fold, about 5-fold to about 30-fold, about 5-fold toabout 20-fold, about 5-fold to about 15-fold, about 5-fold to about10-fold, about 5-fold to about 8-fold, about 5-fold to about 6-fold,about 10-fold to about 100-fold, about 10-fold to about 90-fold, about10-fold to about 80-fold, about 10-fold to about 70-fold, about 10-foldto about 60-fold, about 10-fold to about 50-fold, about 10-fold to about40-fold, about 10-fold to about 30-fold, about 10-fold to about 20-fold,about 10-fold to about 15-fold, about 15-fold to about 100-fold, about15-fold to about 90-fold, about 15-fold to about 80-fold, about 15-foldto about 70-fold, about 15-fold to about 60-fold, about 15-fold to about50-fold, about 15-fold to about 40-fold, about 15-fold to about 30-fold,about 15-fold to about 20-fold, about 20-fold to about 100-fold, about20-fold to about 90-fold, about 20-fold to about 80-fold, about 20-foldto about 70-fold, about 20-fold to about 60-fold, about 20-fold to about50-fold, about 20-fold to about 40-fold, about 20-fold to about 30-fold,about 30-fold to about 40-fold, about 40-fold to about 100-fold, about40-fold to about 90-fold, about 40-fold to about 80-fold, about 40-foldto about 70-fold, about 40-fold to about 60-fold, about 40-fold to about50-fold, about 50-fold to about 100-fold, about 50-fold to about90-fold, about 50-fold to about 80-fold, about 50-fold to about 70-fold,about 50-fold to about 60-fold, about 60-fold to about 100-fold, about60-fold to about 90-fold, about 60-fold to about 80-fold, about 60-foldto about 70-fold, about 70-fold to about 100-fold, about 70-fold toabout 90-fold, about 70-fold to about 80-fold, about 80-fold to about100-fold, about 80-fold to about 90-fold, or about 90-fold to about100-fold) greater than the presence of the analyte at the differentlocation(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., about 1% to about 95%,about 1% to about 90%, about 1% to about 85%, about 1% to about 80%,about 1% to about 75%, about 1% to about 70%, about 1% to about 65%,about 1% to about 60%, about 1% to about 55%, about 1% to about 50%,about 1% to about 45%, about 1% to about 40%, about 1% to about 35%,about 1% to about 30%, about 1% to about 25%, about 1% to about 20%,about 1% to about 15%, about 1% to about 10%, about 1% to about 5%,about 5% to about 99%, about 5% to about 95%, about 5% to about 90%,about 5% to about 85%, about 5% to about 80%, about 5% to about 75%,about 5% to about 70%, about 5% to about 65%, about 5% to about 60%,about 5% to about 55%, about 5% to about 50%, about 5% to about 45%,about 5% to about 40%, about 5% to about 35%, about 5% to about 30%,about 5% to about 25%, about 5% to about 20%, about 5% to about 15%,about 5% to about 10%, about 10% to about 99%, about 10% to about 95%,about 10% to about 90%, about 10% to about 85%, about 10% to about 80%,about 10% to about 75%, about 10% to about 70%, about 10% to about 65%,about 10% to about 60%, about 10% to about 55%, about 10% to about 50%,about 10% to about 45%, about 10% to about 40%, about 10% to about 35%,about 10% to about 30%, about 10% to about 25%, about 10% to about 20%,about 10% to about 15%, about 20% to about 99%, about 20% to about 95%,about 20% to about 90%, about 20% to about 85%, about 20% to about 80%,about 20% to about 75%, about 20% to about 70%, about 20% to about 65%,about 20% to about 60%, about 20% to about 55%, about 20% to about 50%,about 20% to about 45%, about 20% to about 40%, about 20% to about 35%,about 20% to about 30%, about 20% to about 25%, about 30% to about 99%,about 30% to about 95%, about 30% to about 90%, about 30% to about 85%,about 30% to about 80%, about 30% to about 75%, about 30% to about 70%,about 30% to about 65%, about 30% to about 60%, about 30% to about 55%,about 30% to about 50%, about 30% to about 45%, about 30% to about 40%,about 30% to about 35%, about 40% to about 99%, about 40% to about 95%,about 40% to about 90%, about 40% to about 85%, about 40% to about 80%,about 40% to about 75%, about 40% to about 70%, about 40% to about 65%,about 40% to about 60%, about 40% to about 55%, about 40% to about 50%,about 40% to about 45%, about 50% to about 99%, about 50% to about 95%,about 50% to about 90%, about 50% to about 85%, about 50% to about 80%,about 50% to about 75%, about 50% to about 70%, about 50% to about 65%,about 50% to about 60%, about 50% to about 55%, about 60% to about 99%,about 60% to about 95%, about 60% to about 90%, about 60% to about 85%,about 60% to about 80%, about 60% to about 75%, about 60% to about 70%,about 60% to about 65%, about 70% to about 99%, about 70% to about 95%,about 70% to about 90%, about 70% to about 85%, about 70% to about 80%,about 70% to about 75%, about 80% to about 99%, about 80% to about 95%,about 80% to about 90%, about 80% to about 85%, about 90% to about 99%,about 90% to about 95%, or about 95% to about 99%) decreased than thepresence of the analyte at the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of tissue to be resected.

In some embodiments, the method further comprises comparing presence ofone or more additional analyte(s) at the location in the tissue samplewith the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., about 1 to about18,000, about 1 to about 16,000, about 1 to about 14,000, about 1 toabout 12,000, about 1 to about 10,000, about 1 to about 9,000, about 1to about 8,000, about 1 to about 7,000, about 1 to about 6,000, about 1to about 5,000, about 1 to about 4,500, about 1 to about 4,000, about 1to about 3,500, about 1 to about 3,000, about 1 to about 2,500, about 1to about 2,000, about 1 to about 1,500, about 1 to about 1,000, about 1to about 800, about 1 to about 600, about 1 to about 500, about 1 toabout 400, about 1 to about 300, about 1 to about 200, about 1 to about100, about 1 to about 50, about 1 to about 20, about 1 to about 10,about 1 to about 5, about 50 to about 20,000, about 50 to about 18,000,about 50 to about 16,000, about 50 to about 14,000, about 50 to about12,000, about 50 to about 10,000, about 50 to about 9,000, about 50 toabout 8,000, about 50 to about 7,000, about 50 to about 6,000, about 50to about 5,000, about 50 to about 4,500, about 50 to about 4,000, about50 to about 3,500, about 50 to about 3,000, about 50 to about 2,500,about 50 to about 2,000, about 50 to about 1,500, about 50 to about1,000, about 50 to about 800, about 50 to about 600, about 50 to about500, about 50 to about 400, about 50 to about 300, about 50 to about200, about 50 to about 100, about 100 to about 20,000, about 100 toabout 18,000, about 100 to about 16,000, about 100 to about 14,000,about 100 to about 12,000, about 100 to about 10,000, about 100 to about9,000, about 100 to about 8,000, about 100 to about 7,000, about 100 toabout 6,000, about 100 to about 5,000, about 100 to about 4,500, about100 to about 4,000, about 100 to about 3,500, about 100 to about 3,000,about 100 to about 2,500, about 100 to about 2,000, about 100 to about1,500, about 100 to about 1,000, about 100 to about 800, about 100 toabout 600, about 100 to about 500, about 100 to about 400, about 100 toabout 300, about 100 to about 200, about 500 to about 20,000, about 500to about 18,000, about 500 to about 16,000, about 500 to about 14,000,about 500 to about 12,000, about 500 to about 10,000, about 500 to about9,000, about 500 to about 8,000, about 500 to about 7,000, about 500 toabout 6,000, about 500 to about 5,000, about 500 to about 4,500, about500 to about 4,000, about 500 to about 3,500, about 500 to about 3,000,about 500 to about 2,500, about 500 to about 2,000, about 500 to about1,500, about 500 to about 1,000, about 500 to about 800, about 500 toabout 600, about 1,000 to about 20,000, about 1,000 to about 18,000,about 1,000 to about 16,000, about 1,000 to about 14,000, about 1,000 toabout 12,000, about 1,000 to about 10,000, about 1,000 to about 9,000,about 1,000 to about 8,000, about 1,000 to about 7,000, about 1,000 toabout 6,000, about 1,000 to about 5,000, about 1,000 to about 4,500,about 1,000 to about 4,000, about 1,000 to about 3,500, about 1,000 toabout 3,000, about 1,000 to about 2,500, about 1,000 to about 2,000,about 1,000 to about 1,500, about 1,500 to about 20,000, about 2,000 toabout 20,000, about 2,000 to about 18,000, about 2,000 to about 16,000,about 2,000 to about 14,000, about 2,000 to about 12,000, about 2,000 toabout 10,000, about 2,000 to about 9,000, about 2,000 to about 8,000,about 2,000 to about 7,000, about 2,000 to about 6,000, about 2,000 toabout 5,000, about 2,000 to about 4,500, about 2,000 to about 4,000,about 2,000 to about 3,500, about 2,000 to about 3,000, about 2,000 toabout 2,500, about 5,000 to about 20,000, about 5,000 to about 18,000,about 5,000 to about 16,000, about 5,000 to about 14,000, about 5,000 toabout 12,000, about 5,000 to about 10,000, about 5,000 to about 9,000,about 5,000 to about 8,000, about 5,000 to about 7,000, about 5,000 toabout 6,000, about 10,000 to about 20,000, about 10,000 to about 18,000,about 10,000 to about 16,000, about 10,000 to about 14,000, about 10,000to about 12,000, about 12,000 to about 20,000, about 12,000 to about18,000, about 12,000 to about 16,000, about 12,000 to about 14,000,about 14,000 to about 20,000, about 14,000 to about 18,000, about 14,000to about 16,000, about 16,000 to about 20,000, about 16,000 to about18,000, or about 18,000 to about 20,000) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 (e.g., about 1 to about 90,000, about 1 to about80,000, about 1 to about 70,000, about 1 to about 60,000, about 1 toabout 50,000, about 1 to about 40,000, about 1 to about 30,000, about 1to about 20,000, about 1 to about 10,000, about 1 to about 9,000, about1 to about 8,000, about 1 to about 7,000, about 1 to about 6,000, about1 to about 5,000, about 1 to about 4,000, about 1 to about 3,000, about1 to about 2,000, about 1 to about 1,000, about 1 to about 900, about 1to about 800, about 1 to about 700, about 1 to about 600, about 1 toabout 500, about 1 to about 400, about 1 to about 300, about 1 to about200, about 1 to about 100, about 1 to about 90, about 1 to about 80,about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1to about 40, about 1 to about 30, about 1 to about 20, about 1 to about10, about 100 to about 100,000, about 100 to about 90,000, about 100 toabout 80,000, about 100 to about 70,000, about 100 to about 60,000,about 100 to about 50,000, about 100 to about 40,000, about 100 to about30,000, about 100 to about 20,000, about 100 to about 10,000, about 100to about 9,000, about 100 to about 8,000, about 100 to about 7,000,about 100 to about 6,000, about 100 to about 5,000, about 100 to about4,000, about 100 to about 3,000, about 100 to about 2,000, about 100 toabout 1,000, about 100 to about 900, about 100 to about 800, about 100to about 700, about 100 to about 600, about 100 to about 500, about 100to about 400, about 100 to about 300, about 100 to about 200, about1,000 to about 100,000, about 1,000 to about 90,000, about 1,000 toabout 80,000, about 1,000 to about 70,000, about 1,000 to about 60,000,about 1,000 to about 50,000, about 1000 to about 40,000, about 1,000 toabout 30,000, about 1,000 to about 20,000, about 1,000 to about 10,000,about 1000 to about 9,000, about 1,000 to about 8,000, about 1,000 toabout 7,000, about 1000 to about 6,000, about 1,000 to about 5,000,about 1,000 to about 4,000, about 1000 to about 3,000, about 1,000 toabout 2,000, 10,000 to about 100,000, about 10,000 to about 90,000,about 10,000 to about 80,000, about 10,000 to about 70,000, about 10,000to about 60,000, about 10000 to about 50,000, about 10,000 to about40,000, about 10,000 to about 30,000, about 10,000 to about 20,000)cells.

In some embodiments of any one of the methods described herein, thetissue sample can be obtained from any suitable tissue or organ from thesubject (e.g., breast tissue, muscle tissue, gland tissue, fat oradipose tissue, nerve tissue, joint tissue, ligament tissue, tendontissue, mouth tissue, tongue tissue, salivary gland tissue, parotidgland tissue, submandibular gland tissue, sublingual gland tissue,pharynx tissue, esophageal tissue, stomach tissue, small intestinetissue, duodenum tissue, jejunum tissue, ileum tissue, large intestinetissue, liver tissue, gallbladder tissue, mesentery tissue, pancreastissue, anal canal tissue, anus tissue, nasal cavity tissue, pharynxtissue, larynx tissue, trachea tissue, bronchi tissue, lung tissue,diaphragm tissue, kidney tissue, ureter tissue, bladder tissue, urethratissue, ovarian tissue, fallopian tube tissue, uterus tissue, vaginatissue, vulva tissue, clitoris tissue, testes tissue, epididymis tissue,vas deferens tissue, seminal vesicles tissue, prostate tissue,bulbourethral gland tissue, external reproductive organ tissue, penistissue, scrotum tissue, brain tissue, pituitary gland tissue, pinealgland tissue, thyroid gland tissue, parathyroid gland tissue, adrenalgland tissue, pancreas tissue, heart tissue, patent foramen ovaletissue, artery tissue, vein tissue, capillary tissue, lymphatic vesseltissue, lymph node tissue, bone tissue, thymus tissue, spleen tissue,gut-associated lymphoid tissue, tonsil tissue, interstitium tissue,cerebrum tissue, cerebral hemisphere tissue, diencephalon tissue,brainstem tissue, midbrain tissue, pons tissue, medulla oblongatatissue, cerebellum tissue, spinal cord tissue, ventricular systemtissue, choroid plexus tissue, nerve tissue, cranial nerve tissue,spinal nerve tissue, Anglia tissue, enteric nervous system tissue, eyetissue, a cornea tissue, iris tissue, ciliary body tissue, lens tissue,retina tissue, ear tissue, outer ear tissue, earlobe tissue, eardrumtissue, middle ear tissue, ossicles tissue, inner ear tissue, cochleatissue, vestibule tissue, semicircular canal tissue, olfactoryepithelium tissue, tongue tissue, taste bud tissue, mammary glandtissue, skin tissue, subcutaneous tissue, and milk duct tissue). In someembodiments, the tissue sample can include or be proximal in the body ofthe subject to a nerve, a blood vessel, and/or a lymph vessel.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MM, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy from the subject).

In some embodiments of any of the methods described herein, at least aportion of the tissue to be resected includes cancer cell(s),pre-cancerous cell(s), necrotic cell(s), infected cell(s), and/ordiseased tissue. In some embodiments, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, or 100% of the tissue to beresected includes one or more of cancer cell(s), pre-cancerous cell(s),necrotic cell(s), infected cell(s), and disease tissue.

(b) Methods of Treating a Subject by Resecting Tissue

Also provided herein are methods of treating a subject in need thereofthat include: (a) contacting a tissue sample obtained from the subjectto an array comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises (i) a capture domainthat specifically binds to an analyte of the tissue sample and (ii) aspatial barcode; (b) determining (i) all or a part of a nucleic acidsequence corresponding to the analyte specifically bound to the capturedomain or a complement thereof, and (ii) all or a part of a nucleic acidsequence corresponding to the spatial barcode or a complement thereof,and using the determined nucleic acid sequences of (i) and (ii) toidentify the presence of the analyte at a location in the tissue sample;(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at a different location in the tissuesample, and determining the surgical margin based on the comparison; and(d) resecting tissue from the subject using the surgical margindetermined in step (c).

A method of treating a subject, the method comprising: resecting tissuefrom the subject using a surgical margin previously determined using amethod comprising the steps of: (a) contacting a tissue sample obtainedfrom the subject to an array comprising a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises (i)a capture domain that specifically binds to an analyte of the tissuesample and (ii) a spatial barcode; (b) determining (i) all or a part ofa nucleic acid sequence corresponding to the analyte specifically boundto the capture domain or a complement thereof, and (ii) all or a part ofa nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (c) comparing the presence of the analyte at thelocation in the tissue sample to the presence of the analyte at adifferent location in the tissue sample, and determining the surgicalmargin based on the comparison.

For example, when a clinician practices the method, the data obtainedcan provide the clinician with information on the accurate location ofthe cancerous or diseased tissue, therefore provide the accuratesurgical margin of the tissue to be resected. Using the information ofthe accurate surgical margin provided by the method described herein,the clinician is able to achieve, e.g., more complete resection, therebytreating the subject.

In some embodiments, the analyte is a DNA or RNA. In some embodiments,the analyte is a messenger RNA (mRNA) molecule. In some embodiments, theanalyte is a genomic DNA. In some embodiments, the analyte comprises afull-length sequence of a biomarker described herein. In someembodiments, the analyte comprises a fragment of the sequence of abiomarker described herein. In some embodiments of any of the methodsdescribed herein, each of the plurality of capture probes comprises (i)a capture domain that binds specifically to an analyte of the tissuesample and (ii) a spatial barcode. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the sequence of the analyteof the tissue sample. In some embodiments, the capture domain can have atotal of about 10 nucleotides to about 125 nucleotides (or any of thesubranges of this range described herein). In some embodiments, thesequence that is substantially complementary to a portion of thesequence of the analyte can be a random sequence. In some embodiments,the sequence that is substantially complementary to a portion of thesequence of the analyte can include a poly(T) oligonucleotide sequence(e.g., at least 5 contiguous Ts, at least 10 contiguous Ts, or at least15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundcDNA as a template, and generating a single-stranded nucleic acidcomprising a sequence that is complementary to all or a part of theextended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode, or a complement thereof,are described herein or are known in the art.

Also provided herein are methods of treating a subject that include: (a)contacting a tissue sample obtained from the subject to a plurality ofanalyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte; (b) disposing the tissue sample ontoan array, wherein the array comprises a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain that binds specifically to theanalyte capture sequence; (c) determining (i) all or a part of a nucleicacid sequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison; and(e) resecting tissue from the subject using the surgical margindetermined in step (d).

Also provided herein are methods of treating a subject, the methodcomprising: resecting tissue from the subject using a surgical marginpreviously determined using a method comprising the steps of: (a)contacting a tissue sample obtained from the subject to a plurality ofanalyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte; (b) disposing the tissue sample ontoan array, wherein the array comprises a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain that binds specifically to theanalyte capture sequence; (c) determining (i) all or a part of a nucleicacid sequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

In some embodiments, the analyte is a protein. In some embodiments, theanalyte is a full-length protein. In some embodiments, the analyte is afragment of a protein. In some embodiments, the analyte is a byproductof a protein. In some embodiments, the protein is any of the exemplarycancer biomarkers described herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundanalyte capture sequence as a template, and generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe. Non-limiting methods for determiningthe sequence of (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode, or a complementthereof, or (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode, or a complement thereof, aredescribed herein or are known in the art.

In some embodiments, the resected tissue is or comprises a tumor (e.g.,a malignant or a benign tumor). In some embodiments, the tumor is asolid tumor. In some embodiments, the subject is suspected of having acancer. In some embodiments, the subject has been previously diagnosedor identified as having a cancer (e.g., any of the exemplary cancersdescribed herein).

In some embodiments, the resected tissue can include a tumor, (e.g., amalignant tumor) of any of the types of cancer describes herein.

In some embodiments, the analyte is a tumor biomarker. In someembodiments, the analyte is a tumor antigen. Exemplary tumor antigensinclude, but are not limited to, any of the exemplary tumor antigensdescribed herein.

In some embodiments, the resected tissue is or comprises an infectedtissue, a necrotic tissue, or a diseased tissue. In some embodiments,the analyte can be associated with an infection, necrosis, inflammation,or disease. Non-limiting examples of such analytes are known in the art.

In some embodiments, the resected tissue is infected by a bacterium(e.g., any of the exemplary bacteria described herein), a parasite orprotozoa (e.g., any of the exemplary parasites or protozoa describedherein), a fungus (e.g., any of the exemplary fungi described herein),or a virus (e.g., any of the exemplary viruses described herein).

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to thepresence of the analyte at different location(s) in the tissue sample,and determining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., or any of thesubranges of this range described herein) greater than the presence ofthe analyte at the different location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., or any of the subranges ofthis range described herein) decreased than the presence of the analyteat the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of tissue to be resected.

In some embodiments, the method further comprises comparing the presenceof one or more additional analyte(s) at the location in the tissuesample with the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., or any of thesubranges of this range described herein) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 cells.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MM, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy sample from the subject).

In some embodiments of any of the methods described herein, at least aportion of the resected tissue includes cancer cell(s), pre-cancerouscell(s), necrotic cell(s), infected cell(s), and/or diseased tissue. Insome embodiments, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or 100% of the resected tissue includes one or moreof cancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue.

In some embodiments, the tissue resection is considered successful whenless than 20%, less than 15%, less than 10%, less than 5%, or zerocancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and/or diseased tissue are detected post-resection as comparedto the identified cancer cell(s), pre-cancerous cell(s), necroticcell(s), infected cell(s), and/or diseased tissue prior to theresection.

In some embodiments, the treatment is considered successful when lessthan 20%, less than 15%, less than 10%, less than 5%, or zero cancercell(s), pre-cancerous cell(s), necrotic cell(s), infected cell(s),and/or diseased tissue are detected post-resection as compared to theidentified cancer cell(s), pre-cancerous cell(s), necrotic cell(s),infected cell(s), and/or diseased tissue prior to the resection.

In some embodiments of any of the methods described herein, the surgicalmargin can be the margin between the location(s) of one or more ofcancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue, and the location(s) of healthy or normaltissue, in a subject.

(c) Methods of Identifying a Surgical Margin of a Tissue to be Resected

Provided herein are methods of identifying a surgical margin of a tissueto be resected in a subject, the method comprising: (a) contacting atissue sample obtained from the subject to an array comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises (i) a capture domain that specifically binds toan analyte of the tissue sample and (ii) a spatial barcode; (b)determining (i) all or a part of the nucleic acid sequence correspondingto the analyte specifically bound to the capture domain or a complementthereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (c)comparing the presence of the analyte at the location in the tissuesample to the presence of the analyte at different location(s) in thetissue sample, and determining a surgical margin of a tissue to beresected from the subject based on the comparison. In some embodiments,the analyte is a DNA or RNA. In some embodiments, the analyte is amessenger RNA (mRNA) molecule. In some embodiments, the analyte is agenomic DNA. In some embodiments, the analyte comprises a full-lengthsequence of a biomarker described herein. In some embodiments, theanalyte comprises a fragment of the sequence of a biomarker describedherein. In some embodiments of any of the methods described herein, eachof the plurality of capture probes comprises (i) a capture domain thatbinds specifically to an analyte of the tissue sample and (ii) a spatialbarcode. The capture probe can be any capture probe described herein. Insome embodiments, the capture domain of the capture probe comprises asequence that is substantially complementary (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100%complementary) to a portion of the sequence of the analyte of the tissuesample. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the sequence of theanalyte can be a random sequence. In some embodiments, the sequence thatis substantially complementary to a portion of the sequence of theanalyte can include a poly(T) oligonucleotide sequence (e.g., at least 5contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundcDNA as a template, and generating a single-stranded nucleic acidcomprising a sequence that is complementary to all or a part of theextended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode, or a complement thereof,are described herein or are known in the art.

Provided herein are methods of identifying a surgical margin of a tissueto be resected in a subject that include: (a) contacting a tissue sampleobtained from the subject to a plurality of analyte capture agents,where an analyte capture agent of the plurality of analyte captureagents comprises an analyte binding moiety barcode, an analyte capturesequence, and an analyte binding moiety that binds specifically to ananalyte; (b) disposing the tissue sample onto an array, wherein thearray comprises a plurality of capture probes, where a capture probe ofthe plurality of capture probes comprises a spatial barcode and acapture domain that binds specifically to the analyte capture sequence;(c) determining (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing the the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining a surgical margin of a tissue to be resectedfrom the subject based on the comparison. In some embodiments, theanalyte is a protein. In some embodiments, the analyte is a full-lengthprotein. In some embodiments, the analyte is a fragment of a protein. Insome embodiments, the analyte is a byproduct of a protein. In someembodiments, the protein is any of the exemplary cancer biomarkersdescribed herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, sequencing by ligation or any of the othermethods for sequencing described herein or known in the art. Forexample, sequencing can involve one or more of nucleic acidamplification, the ligation or addition of one or more sequencingadaptors, cleavage of the capture probe from the array, extension of thecapture probe using the bound analyte capture sequence as a template,and generating a single-stranded nucleic acid comprising a sequence thatis complementary to all or a part of the extended capture probe.Non-limiting methods for determining the sequence of (i) all or a partof the nucleic acid sequence corresponding to the analyte binding moietybarcode, or a complement thereof, or (ii) all or a part of the nucleicacid sequence corresponding to the spatial barcode, or a complementthereof, are described herein or are known in the art.

In some embodiments, the tissue to be resected is or comprises a tumor(e.g., a malignant or a benign tumor). In some embodiments, the tumor isa solid tumor. In some embodiments, the subject is suspected of having acancer. In some embodiments, the subject has been previously diagnosedor identified as having a cancer (e.g., any of the exemplary cancersdescribed herein).

In some embodiments, the tissue to be resected can include a tumor(e.g., a malignant tumor) of any of the types of cancer describesherein.

In some embodiments, the analyte is a tumor biomarker. In someembodiments, the analyte is a tumor antigen. Exemplary tumor antigensinclude, but are not limited to, any of the exemplary tumor antigensdescribed herein.

In some embodiments, the tissue to be resected is or comprises aninfected tissue, a necrotic tissue, or a diseased tissue. In someembodiments, the analyte can be associated with an infection, necrosis,inflammation, or disease. Non-limiting examples of such analytes areknown in the art.

In some embodiments, the tissue to be resected is infected by abacterium (e.g., any of the exemplary bacteria described herein), aparasite or protozoa (e.g., any of the exemplary parasites or protozoadescribed herein), a fungus (e.g., any of the exemplary fungi describedherein), or a virus (e.g., any of the exemplary viruses describedherein).

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to thepresence of the analyte at different location(s) in the tissue sample,and determining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., or any of thesubranges of this range described herein) greater than the presence ofthe analyte at the different location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., or any of the subranges ofthis range described herein) decreased than the presence of the analyteat the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of tissue to be resected.

In some embodiments, the method further comprises comparing presence ofone or more additional analyte(s) at the location in the tissue samplewith the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., or any of thesubranges of this range described herein) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 cells.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MRI, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy sample from the subject).

In some embodiments of any of the methods described herein, at least aportion of the tissue to be resected includes cancer cell(s),pre-cancerous cell(s), necrotic cell(s), infected cell(s), and/ordiseased tissue. In some embodiments, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, or 100% of the tissue to beresected includes one or more of cancer cell(s), pre-cancerous cell(s),necrotic cell(s), infected cell(s), and disease tissue.

In some embodiments, the tissue resection is considered successful whenless than 20%, less than 15%, less than 10%, less than 5%, or zerocancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and/or diseased tissue are detected post-resection as comparedto the identified cancer cell(s), pre-cancerous cell(s), necroticcell(s), infected cell(s), and/or diseased tissue prior to theresection.

In some embodiments of any of the methods described herein, the surgicalmargin can be the margin between the location(s) of one or more ofcancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue, and the location(s) of healthy or normaltissue, in a subject.

(d) Methods of Reducing the Risk of Re-Excision of a Tissue

Also provided herein are methods of reducing the risk of re-excision ofa tissue from a subject that include: (a) contacting a tissue sampleobtained from the subject to an array comprising a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises a (i) capture domain that specifically binds to an analyte ofthe tissue sample and (ii) a spatial barcode; (b) determining (i) all ora part of a nucleic acid sequence corresponding to the analytespecifically bound to the capture domain or a complement thereof, and(ii) all or a part of a nucleic acid sequence corresponding to thespatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (c) comparing the presenceof the analyte at the location in the tissue sample to the presence ofthe analyte at different location(s) in the tissue sample, anddetermining the surgical margin based on the comparison and (d)resecting tissue from the subject using the surgical margin determinedin step (c), wherein the method results in a reduction in the risk offuture re-excision of the tissue in the subject.

Also provided herein are methods of reducing the risk of re-excision ofa tissue from a subject, the method comprising: resecting tissue fromthe subject using a surgical margin previously determined using a methodcomprising the steps of: (a) contacting a tissue sample obtained fromthe subject to an array comprising a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises a(i) capture domain that specifically binds to an analyte of the tissuesample and (ii) a spatial barcode; (b) determining (i) all or a part ofa nucleic acid sequence corresponding to the analyte specifically boundto the capture domain or a complement thereof, and (ii) all or a part ofa nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (c) comparing the presence of the analyte at thelocation in the tissue sample to presence of the analyte at differentlocation(s) in the tissue sample, and determining the surgical marginbased on the comparison.

For example, when a clinician practices the method, the data obtainedcan provide the clinician with guidance on where to resect the tissuemargins thereby reducing the probability that an additional resectionmight be needed; the clinician will be more confident in capturingtissue margins for a more complete resection. In some embodiments, theanalyte is a DNA or RNA. In some embodiments, the analyte is a messengerRNA (mRNA) molecule. In some embodiments, the analyte is a genomic DNA.In some embodiments, the analyte comprises a full-length sequence of abiomarker described herein. In some embodiments, the analyte comprises afragment of the sequence of a biomarker described herein. In someembodiments of any of the methods described herein, each of theplurality of capture probes comprises (i) a capture domain that bindsspecifically to an analyte of the tissue sample and (ii) a spatialbarcode. The capture probe can be any capture probe described herein. Insome embodiments, the capture domain of the capture probe comprises asequence that is substantially complementary (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100%complementary) to a portion of the sequence of the analyte of the tissuesample. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the sequence of theanalyte can be a random sequence. In some embodiments, the sequence thatis substantially complementary to a portion of the sequence of theanalyte can include a poly(T) oligonucleotide sequence (e.g., at least 5contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundcDNA as a template, and generating a single-stranded nucleic acidcomprising a sequence that is complementary to all or a part of theextended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode, or a complement thereof,are described herein or are known in the art.

Also provided herein are methods of reducing the risk of re-excision ofa tissue from a subject that include: (a) contacting a tissue sampleobtained from the subject to a plurality of analyte capture agents,wherein an analyte capture agent of the plurality of analyte captureagents comprises an analyte binding moiety barcode, an analyte capturesequence, and an analyte binding moiety that binds specifically to ananalyte; (b) disposing the tissue sample onto an array, wherein thearray comprises a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises a spatial barcode and acapture domain that binds specifically to the analyte capture sequence;(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (d) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin based on the comparison; and (e) resecting tissuefrom the subject using the surgical margin determined in step (d), wherethe method results in a reduction in the risk of future re-excision ofthe tissue in the subject. In some embodiments, the analyte is aprotein.

Also provided herein are methods of reducing the risk of re-excision ofa tissue from a subject, the method comprising: resecting tissue fromthe subject using a surgical margin previously determined using a methodcomprising the steps of: (a) contacting a tissue sample obtained fromthe subject to a plurality of analyte capture agents, wherein an analytecapture agent of the plurality of analyte capture agents comprises ananalyte binding moiety barcode, an analyte capture sequence, and ananalyte binding moiety that binds specifically to an analyte; (b)disposing the tissue sample onto an array, wherein the array comprises aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain thatbinds specifically to the analyte capture sequence; (c) determining (i)all or a part of a nucleic acid sequence corresponding to the analytebinding moiety barcode or a complement thereof, and (ii) all or a partof a nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (d) comparing the presence of the analyte at thelocation in the tissue sample to the presence of the analyte atdifferent location(s) in the tissue sample, and determining the surgicalmargin based on the comparison.

In some embodiments, the analyte is a full-length protein. In someembodiments, the analyte is a fragment of a protein. In someembodiments, the analyte is a byproduct of a protein. In someembodiments, the protein is any of the exemplary cancer biomarkersdescribed herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundanalyte capture sequence as a template, and generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe. Non-limiting methods for determiningthe sequence of (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode, or a complementthereof, or (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode, or a complement thereof, aredescribed herein or are known in the art.

In some embodiments, the resected tissue is or comprises a tumor (e.g.,a malignant or a benign tumor). In some embodiments, the tumor is asolid tumor. In some embodiments, the subject is suspected of having acancer. In some embodiments, the subject has been previously diagnosedor identified as having a cancer (e.g., any of the exemplary cancersdescribed herein).

In some embodiments, the resected tissue can include a tumor (e.g., amalignant tumor) of any of the types of cancer describes herein.

In some embodiments, the analyte is a tumor biomarker. In someembodiments, the analyte is a tumor antigen. Exemplary tumor antigensinclude, but are not limited to, any of the exemplary tumor antigensdescribed herein.

In some embodiments, the resected tissue is or comprises an infectedtissue, a necrotic tissue, or a diseased tissue. In some embodiments,the analyte can be associated with an infection, necrosis, inflammation,or disease. Non-limiting examples of such analytes are known in the art.

In some embodiments, the resected tissue is infected by a bacterium(e.g., any of the exemplary bacteria described herein), a parasite orprotozoa (e.g., any of the exemplary parasites or protozoa describedherein), a fungus (e.g., any of the exemplary fungi described herein),or a virus (e.g., any of the exemplary viruses described herein).

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to presenceof the analyte at different location(s) in the tissue sample, anddetermining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., or any of thesubranges of this range described herein) greater than the presence ofthe analyte at the different location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., or any of the subranges ofthis range described herein) decreased than the presence of the analyteat the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of tissue to be resected.

In some embodiments, the method further comprises comparing the presenceof one or more additional analyte(s) at the location in the tissuesample with the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., or any of thesubranges of this range described herein) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 cells.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MM, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy sample from the subject).

In some embodiments of any of the methods described herein, at least aportion of the resected tissue includes cancer cell(s), pre-cancerouscell(s), necrotic cell(s), infected cell(s), and/or diseased tissue. Insome embodiments, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or 100% of the resected tissue includes one or moreof cancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue.

In some embodiments of any of the methods described herein, the surgicalmargin can be the margin between the location(s) of one or more ofcancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue, and the location(s) of healthy or normaltissue, in a subject.

In some embodiments, the methods described herein are more accurate indetermining the surgical margin of a tissue to be resected than thetraditional methods, such as medical imaging or scanning methods.

In some embodiments, a re-excision includes additional tissue excisionsduring the initial surgical procedure to obtain the tissue sample. Insome embodiments, a re-excision includes additional tissue excisionsduring a future procedure.

In some embodiments, the methods described herein results in a reduction(e.g., at least a 5% reduction, at least a 10% reduction, at least a 15%reduction, at least a 20% reduction, at least a 25% reduction, at leasta 30% reduction, at least a 35% reduction, at least a 40% reduction, atleast a 45% reduction, at least a 50% reduction, at least a 55%reduction, at least a 60% reduction, at least a 65% reduction, at leasta 70% reduction, at least a 75% reduction, at least a 80% reduction, atleast a 85% reduction, at least a 90% reduction, at least a 95%reduction, or at least a 99% reduction, or about a 1% to about a 5%reduction, about a 5% to about a 10% reduction, about a 10% to about a99% reduction, about a 10% to about a 90% reduction, about a 10% toabout a 80% reduction, about a 10% to about a 70% reduction, about a 10%to about a 60% reduction, about a 10% to about a 50% reduction, about a10% to about a 40% reduction, about a 10% to about a 30% reduction,about a 10% to about a 20% reduction, about a 10% to about a 15%reduction, about a 20% to about a 99% reduction, about a 20% to about a90% reduction, about a 20% to about a 80% reduction, about a 20% toabout a 70% reduction, about a 20% to about a 60% reduction, about a 20%to about a 50% reduction, about a 20% to about a 40% reduction, about a20% to about a 30% reduction, about a 30% to about a 99% reduction,about a 30% to about a 90% reduction, about a 30% to about a 80%reduction, about a 30% to about a 70% reduction, about a 30% to about a60% reduction, about a 30% to about a 50% reduction, about a 30% toabout a 40% reduction, about a 40% to about a 99% reduction, about a 40%to about a 90% reduction, about a 40% to about a 80% reduction, about a40% to about a 70% reduction, about a 40% to about a 60% reduction,about a 40% to about a 50% reduction, about a 50% to about a 99%reduction, about a 50% to about a 90% reduction, about a 50% to about a50% to about a 80% reduction, about a 50% to about a 70% reduction,about a 50% to about a 65% reduction, about a 50% to about a 60%reduction, about a 50% to about a 55% reduction, about a 60% to about a99% reduction, about a 60% to about a 90% reduction, about a 60% toabout a 80% reduction, about a 60% to about a 75% reduction, about a 60%to about a 70% reduction, about a 60% to about a 65% reduction, about a70% to about a 99% reduction, about a 70% to about a 95% reduction,about a 70% to about a 90% reduction, about a 70% to about a 85%reduction, about a 70% to about a 80% reduction, about a 70% to about a75% reduction, about a 80% to about a 99% reduction, about a 80% toabout a 95% reduction, about a 80% to about a 90% reduction, about a 80%to about a 85% reduction, about a 90% to about a 99% reduction, about a90% to about a 95% reduction, or about a 95% to about a 99% reduction)in the risk of future re-excision of the tissue in the subject (e.g., asa compared to a similar subject that has undergone resection based on animaging method or by the physician's visual assessment duringresection).

A non-limiting example of a method for identifying a surgical margin ofa tissue to be resected is depicted in FIG. 4. Briefly, a biopsy tissueis excised from a subject and imaged for potential tumor tissue cells orother disease related cells. After imaging, the tissue is sectioned andone or more sections of tissue are subjected to the methods describedherein for spatial determination and location of cells of interest in atissue. For example, gene expression along the margins of a tissuesection indicative of cancer or a disease state and/or the location ofreceptors along the margins of a tissue section indicative of cancer ora disease state is determined via a spatial array. Additionally, knownmutations associated with a cancer or disease state can be spatiallyidentified along the margins of a tissue section. Gene expressionanalysis and/or receptor presence and/or mutational state of cellswithin the tissue margins can be used to determine whether a surgeon hassufficiently resected the tissue. For example, the presence of one ormore genes, receptors and/or mutations indicative of a cancer or diseasestate in a spatially analyzed tissue margin section would indicate thatthe resection of the cancerous or diseased tissue was not complete assuch a more expanded resection might be necessary. When the tissuesection margins are absent of those biomarkers that were used toindicate a cancer or disease state, then a resection could be consideredsuccessful for that location.

(e) Methods of Reducing the Rate of Recurrence of a Tissue Abnormality

Provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject that include: (a) contacting a tissuesample obtained from the subject to an array comprising a plurality ofcapture probes, wherein a capture probe of the plurality of captureprobes comprises a (i) capture domain that specifically binds to ananalyte of the tissue sample and (ii) a spatial barcode; (b) determining(i) all or a part of a nucleic acid sequence corresponding to theanalyte specifically bound to the capture domain or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (c) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin based on the comparison; (d) resecting tissue fromthe subject using the surgical margin determined in step (c), whereinthe method results in a reduction in the rate of recurrence of a tissueabnormality in the subject.

Also provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject, the method comprising: resecting tissuefrom the subject using a surgical margin previously determined using amethod comprising the steps of: (a) contacting a tissue sample obtainedfrom the subject to an array comprising a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises a(i) capture domain that specifically binds to an analyte of the tissuesample and (ii) a spatial barcode; (b) determining (i) all or a part ofa nucleic acid sequence corresponding to the analyte specifically boundto the capture domain or a complement thereof, and (ii) all or a part ofa nucleic acid sequence corresponding to the spatial barcode or acomplement thereof, and using the determined nucleic acid sequences of(i) and (ii) to identify the presence of the analyte at a location inthe tissue sample; (c) comparing the presence of the analyte at thelocation in the tissue sample to the presence of the analyte atdifferent location(s) in the tissue sample, and determining the surgicalmargin based on the comparison.

For example, the methods described herein allow a clinician to identifytissue tumor margins with confidence such that tissue resection willincrease the likelihood that the complete tumor or tissue abnormalityhas been removed from the subject, thereby reducing the rate ofrecurrence of the abnormal tissue or tumor in the subject.

In some embodiments, the analyte is a DNA or RNA. In some embodiments,the analyte is a messenger RNA (mRNA) molecule. In some embodiments, theanalyte is a genomic DNA. In some embodiments, the analyte comprises afull-length sequence of a biomarker described herein. In someembodiments, the analyte comprises a fragment of the sequence of abiomarker described herein. In some embodiments of any of the methodsdescribed herein, each of the plurality of capture probes comprises (i)a capture domain that binds specifically to an analyte of the tissuesample and (ii) a spatial barcode. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the sequence of the analyteof the tissue sample. In some embodiments, the capture domain can have atotal of about 10 nucleotides to about 125 nucleotides (or any of thesubranges of this range described herein). In some embodiments, thesequence that is substantially complementary to a portion of thesequence of the analyte can be a random sequence. In some embodiments,the sequence that is substantially complementary to a portion of thesequence of the analyte can include a poly(T) oligonucleotide sequence(e.g., at least 5 contiguous Ts, at least 10 contiguous Ts, or at least15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundcDNA as a template, and generating a single-stranded nucleic acidcomprising a sequence that is complementary to all or a part of theextended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode, or a complement thereof,are described herein or are known in the art.

Also provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject that include: (a) contacting a tissuesample obtained from the subject to a plurality of analyte captureagents, wherein an analyte capture agent of the plurality of analytecapture agents comprises an analyte binding moiety barcode, an analytecapture sequence, and an analyte binding moiety that binds specificallyto an analyte; (b) disposing the tissue sample onto an array, whereinthe array comprises a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises a spatial barcode anda capture domain that binds specifically to the analyte capturesequence; (c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (d) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin based on the comparison; and (e) resecting tissuefrom the subject using the surgical margin determined in step (d), wherethe method results in a reduction in the rate of recurrence of a tissueabnormality in the subject.

Also provided herein are methods of reducing the rate of recurrence of atissue abnormality in a subject, the method comprising: resecting tissuefrom the subject using a surgical margin previously determined using amethod comprising the steps of: (a) contacting a tissue sample obtainedfrom the subject to a plurality of analyte capture agents, wherein ananalyte capture agent of the plurality of analyte capture agentscomprises an analyte binding moiety barcode, an analyte capturesequence, and an analyte binding moiety that binds specifically to ananalyte; (b) disposing the tissue sample onto an array, wherein thearray comprises a plurality of capture probes, wherein a capture probeof the plurality of capture probes comprises a spatial barcode and acapture domain that binds specifically to the analyte capture sequence;(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample; (d) comparing the presenceof the analyte at the location in the tissue sample to presence of theanalyte at different location(s) in the tissue sample, and determiningthe surgical margin based on the comparison.

In some embodiments, the analyte is a protein. In some embodiments, theanalyte is a full-length protein. In some embodiments, the analyte is afragment of a protein. In some embodiments, the analyte is a byproductof a protein. In some embodiments, the protein is any of the exemplarycancer biomarkers described herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundanalyte capture sequence as a template, and generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe. Non-limiting methods for determiningthe sequence of (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode, or a complementthereof, or (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode, or a complement thereof, aredescribed herein or are known in the art.

In some embodiments, an abnormality in the resected tissue is orcomprises a tumor (e.g., a malignant or a benign tumor). In someembodiments, the tumor is a solid tumor. In some embodiments, thesubject is suspected of having a cancer. In some embodiments, thesubject has been previously diagnosed or identified as having a cancer(e.g., any of the exemplary cancers described herein).

In some embodiments, the resected tissue can include a tumor, e.g., atumor (e.g., a malignant tumor) of any of the types of cancer describesherein.

In some embodiments, the analyte is a tumor biomarker. In someembodiments, the analyte is a tumor antigen. Exemplary tumor antigensinclude, but are not limited to, any of the exemplary tumor antigensdescribed herein.

In some embodiments, the resected tissue is or comprises an infectedtissue, a necrotic tissue, or a diseased tissue. In some embodiments,the analyte can be associated with an infection, necrosis, inflammation,or disease. Non-limiting examples of such analytes are known in the art.

In some embodiments, the resected tissue is infected by a bacterium(e.g., any of the exemplary bacteria described herein), a parasite orprotozoa (e.g., any of the exemplary parasites or protozoa describedherein), a fungus (e.g., any of the exemplary fungi described herein),or a virus (e.g., any of the exemplary viruses described herein).

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to presenceof the analyte at different location(s) in the tissue sample, anddetermining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., or any of thesubranges of this range described herein) greater than the presence ofthe analyte at the different location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., or any of the subranges ofthis range described herein) decreased than the presence of the analyteat the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of tissue to be resected.

In some embodiments, the method further comprises comparing presence ofone or more additional analyte(s) at the location in the tissue samplewith the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., or any of thesubranges of this range described herein) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 cells.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MM, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy sample from the subject).

In some embodiments of any of the methods described herein, at least aportion of the resected tissue includes cancer cell(s), pre-cancerouscell(s), necrotic cell(s), infected cell(s), and/or diseased tissue. Insome embodiments, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or 100% of the resected tissue includes one or moreof cancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue.

In some embodiments of any of the methods described herein, the surgicalmargin can be the margin between the location(s) of one or more ofcancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue, and the location(s) of healthy or normaltissue, in a subject.

In some embodiments, the methods described herein results in a reduction(e.g., at least a 5% reduction, at least a 10% reduction, at least a 15%reduction, at least a 20% reduction, at least a 25% reduction, at leasta 30% reduction, at least a 35% reduction, at least a 40% reduction, atleast a 45% reduction, at least a 50% reduction, at least a 55%reduction, at least a 60% reduction, at least a 65% reduction, at leasta 70% reduction, at least a 75% reduction, at least a 80% reduction, atleast a 85% reduction, at least a 90% reduction, at least a 95%reduction, or at least a 99% reduction, or about a 1% to about a 99%reduction (e.g., or any of the subranges of this range described herein)in the rate of recurrence of a tissue abnormality (e.g., any of thecancers described herein) in the subject (e.g., as a compared to asimilar subject that has undergone resection based on an imaging methodor by the physician's visual assessment during resection).

q(f) Methods of Treating a Subject by Excising or Avoiding Excision of aNerve or Blood Vessel

Also provided herein are methods of treating a subject that include: (a)contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode; (b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (c)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison; and(d) excising a nerve or blood vessel that is within the determinedsurgical margin in the subject, or avoiding excision of a nerve or bloodvessel outside of the determined surgical margin in the subject.

Also provided herein are methods of treating a subject, the methodcomprising: excising a nerve or blood vessel that is within a surgicalmargin in the subject, or avoiding excision of a nerve or blood vesseloutside of the surgical margin in the subject, wherein the surgicalmargin was previously determined using a method comprising the steps of:(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode; (b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (c)comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

For example, when a clinician practices the method, the data obtainedcan provide the clinician with information of the accurate location ofthe cancerous or diseased tissue, therefore provide the accuratesurgical margin of the tissue to be resected. Using the information ofthe accurate surgical margin provided by the method described herein,the clinician is able to achieve, e.g., more complete and accurateexcision, thereby treating the subject in need thereof.

In some embodiments, the analyte is a DNA or RNA. In some embodiments,the analyte is a messenger RNA (mRNA) molecule. In some embodiments, theanalyte is a genomic DNA. In some embodiments, the analyte comprises afull-length sequence of a biomarker described herein. In someembodiments, the analyte comprises a fragment of the sequence of abiomarker described herein. In some embodiments of any of the methodsdescribed herein, each of the plurality of capture probes comprises (i)a capture domain that binds specifically to an analyte of the tissuesample and (ii) a spatial barcode. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the sequence of the analyteof the tissue sample. In some embodiments, the capture domain can have atotal of about 10 nucleotides to about 125 nucleotides (or any of thesubranges of this range described herein). In some embodiments, thesequence that is substantially complementary to a portion of thesequence of the analyte can be a random sequence. In some embodiments,the sequence that is substantially complementary to a portion of thesequence of the analyte can include a poly(T) oligonucleotide sequence(e.g., at least 5 contiguous Ts, at least 10 contiguous Ts, or at least15 contiguous Ts).

In some embodiments of any of the methods described herein, step (b)comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundcDNA as a template, and generating a single-stranded nucleic acidcomprising a sequence that is complementary to all or a part of theextended capture probe. Non-limiting methods for determining thesequence of (i) all or a part of the sequence of the target nucleicacid, or a complement thereof, or (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode, or a complement thereof,are described herein or are known in the art.

Also provided herein are methods of treating a subject that include: (a)contacting a tissue sample obtained from the subject to a plurality ofanalyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte; (b) disposing the tissue sample ontoan array, wherein the array comprises a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain that binds specifically to theanalyte capture sequence; (c) determining (i) all or a part of a nucleicacid sequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing presence of the analyte at the location in the tissue sampleto presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison; and(e) excising a nerve or blood vessel that is within the determinedsurgical margin in the subject, or avoiding excision of a nerve or bloodvessel outside of the determined surgical margin in the subject.

Also provided herein are methods of treating a subject, the methodcomprising: excising a nerve or blood vessel that is within a surgicalmargin in the subject, or avoiding excision of a nerve or blood vesseloutside of the surgical margin in the subject, wherein the surgicalmargin was previously determined using a method comprising the steps of:(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte; (b) disposing the tissue sample ontoan array, wherein the array comprises a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain that binds specifically to theanalyte capture sequence; (c) determining (i) all or a part of a nucleicacid sequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample; (d)comparing presence of the analyte at the location in the tissue sampleto presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

In some embodiments, the analyte is a protein. In some embodiments, theanalyte is a full-length protein. In some embodiments, the analyte is afragment of a protein. In some embodiments, the analyte is a byproductof a protein. In some embodiments, the protein is any of the exemplarycancer biomarkers described herein.

In some embodiments of any of the methods described herein, each of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte. In some embodiments, the analytebinding moiety is an antibody or an antigen-binding antibody fragment(e.g., a Fab). Any other suitable protein binding moiety known in theart can also be used as an analyte binding moiety. In some embodiments,the analyte binding moiety barcode can be any barcode described herein.In some embodiments, the analyte capture sequence can be any analytecapture sequence described herein. In some embodiments of any of themethods described herein, each of the plurality of capture probescomprises a spatial barcode and a capture domain that binds specificallyto the analyte capture sequence. The capture probe can be any captureprobe described herein. In some embodiments, the capture domain of thecapture probe comprises a sequence that is substantially complementary(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% complementary) to a portion of the analyte capturesequence. In some embodiments, the capture domain can have a total ofabout 10 nucleotides to about 125 nucleotides (or any of the subrangesof this range described herein). In some embodiments, the sequence thatis substantially complementary to a portion of the analyte capturesequence can be a random sequence. In some embodiments, the sequencethat is substantially complementary to a portion of the analyte capturesequence can include a poly(T) oligonucleotide sequence (e.g., at least5 contiguous Ts, at least 10 contiguous Ts, or at least 15 contiguousTs).

In some embodiments, the determining of the sequence is by sequencing.In some embodiments, the sequencing is high throughput sequencing,sequencing by hybridization, or any of the other methods for sequencingdescribed herein or known in the art. For example, sequencing caninvolve one or more of nucleic acid amplification, the ligation oraddition of one or more sequencing adaptors, cleavage of the captureprobe from the array, extension of the capture probe using the boundanalyte capture sequence as a template, and generating a single-strandednucleic acid comprising a sequence that is complementary to all or apart of the extended capture probe. Non-limiting methods for determiningthe sequence of (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode, or a complementthereof, or (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode, or a complement thereof, aredescribed herein or are known in the art.

In some embodiments, the tissue to be resected is or comprises a tumor(e.g., a malignant or a benign tumor). In some embodiments, the tumor isa solid tumor. In some embodiments, the subject is suspected of having acancer. In some embodiments, the subject has been previously diagnosedor identified as having a cancer (e.g., any of the exemplary cancersdescribed herein).

In some embodiments, the tissue to be resected can include a tumor(e.g., a malignant tumor) of any of the types of cancer describesherein.

In some embodiments, the analyte is a tumor biomarker. In someembodiments, the analyte is a tumor antigen. Exemplary tumor antigensinclude, but are not limited to, any of the exemplary tumor antigensdescribed herein.

In some embodiments, the tissue to be resected is or comprises aninfected tissue, a necrotic tissue, or a diseased tissue. In someembodiments, the analyte can be associated with an infection, necrosis,inflammation, or disease. Non-limiting examples of such analytes areknown in the art.

In some embodiments, the tissue to be resected is infected by abacterium (e.g., any of the exemplary bacteria described herein), aparasite or protozoa (e.g., any of the exemplary parasites or protozoadescribed herein), a fungus (e.g., any of the exemplary fungi describedherein), or a virus (e.g., any of the exemplary viruses describedherein).

In some embodiments, the methods provided herein comprise comparing thepresence of the analyte at the location in the tissue sample to presenceof the analyte at different location(s) in the tissue sample, anddetermining the size and site of a tissue to be resected from thesubject based on the comparison. In some embodiments, the differentlocation(s) in the tissue sample are reference location(s). In someembodiments, the reference location(s) in the tissue sample arelocations of healthy tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations of non-cancerous tissue.In some embodiments, the reference location(s) in the tissue sample arelocations of non-tumor tissue. In some embodiments, the referencelocation(s) in the tissue sample are locations with no abnormalitiessuch as tumor, cancer, necrosis, inflammation, infection, or disease.

In some embodiments, the presence of the analyte at the location in thetissue sample is significantly different from the presence of theanalyte at the different location(s) in the tissue. In some embodiments,the presence of the analyte at the location in the tissue sample issignificantly greater than the presence of the analyte at the differentlocation(s) in the tissue sample. In some embodiments, the presence ofthe analyte at the location in the tissue sample is significantly lessthan the presence of the analyte at the different location(s) in thetissue sample.

In some embodiments, the location at the tissue sample is determined tobe resected if the presence of the analyte at the location in the tissuesample is significantly different from the presence of the analyte atthe different location(s). In some embodiments, the location at thetissue sample is determined to be resected if the presence of theanalyte at the location in the tissue sample is significantly greaterthan the presence of the analyte at the different location(s). In someembodiments, the location at the tissue sample is determined to beresected if the presence of the analyte at the location in the tissuesample is significantly less than the presence of the analyte at thedifferent location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 0.1-fold to about 100-fold (e.g., or any of thesubranges of this range described herein) greater than the presence ofthe analyte at the different location(s).

In some embodiments, the presence of the analyte at the location in thetissue sample is about 1% to about 99% (e.g., or any of the subranges ofthis range described herein) decreased than the presence of the analyteat the different location(s).

In some embodiments, the presence of certain biomarkers associated witha cancer and/or disease (e.g., breast cancer biomarkers in ductalcarcinoma) at a location in a tissue sample are evaluated. If thepresence of certain biomarkers associated with a cancer and/or diseaseare below a threshold value for those biomarkers, the location in thetissue sample is considered “clear.” If the presence of certainbiomarkers associated with a cancer and/or disease are above a thresholdvalue for those biomarkers, the location in the tissue sample isconsidered within the margin of nerve and/or blood vessel to beresected.

In some embodiments, the method further comprises comparing presence ofone or more additional analyte(s) at the location in the tissue samplewith the presence of the one or more additional analyte(s) at thedifferent location(s) in the tissue sample. In some embodiments, thepresence of a total of about 1 to about 20,000 (e.g., or any of thesubranges of this range described herein) analyte(s) at the location arecompared to the presence of the analyte(s) at the different location(s).

In some embodiments of any one of the methods described herein, mutantcells are identified according to the presence of the one or moreanalyte(s) at the location in the tissue sample. In some embodiments, amutant cell is identified according to the presence of one or morebiomarkers described herein. In some embodiments, a mutant cell isidentified according to the presence of one or more cell-surfacebiomarkers, e.g., a cell-surface receptor, at the location in the tissuesample.

In some embodiments, a cell within a location at the tissue sample isidentified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlydifferent from the presence of the analyte(s) at the differentlocation(s). In some embodiments, a cell within a location at the tissuesample is identified as a mutant cell if the presence of the one or moreanalyte(s) at the location in the tissue sample are significantlygreater than the presence of the analyte(s) at the differentlocation(s). In some embodiments, the mutant cell(s) within the locationat the tissue sample is determined to be resected if the presence of theanalyte(s) at the location in the tissue sample are significantly lessthan the presence of the analyte at the different location(s).

In some embodiments, a location at the tissue sample comprises about 1to about 100,000 cells.

The spatial barcode of the capture probe can be any spatial barcodedescribed herein.

In some embodiments of any of the methods described herein, the arraycan be any of the types of arrays described herein. For example, thearray comprises a slide. In some embodiments, the capture probe isattached to the slide (e.g., by its 5′ end).

In some embodiments of any of the methods described herein, the array isa bead array. In some embodiments, a 5′ end of the capture probe isattached to a bead of the bead array.

In some embodiments of any of the methods described herein, the methodcomprises extending a 3′ end of the capture probe using the specificallybound analyte or analyte binding agent barcode as a template to generatean extended capture probe.

In some embodiments, additional methods are used in combination with themethods described herein to determine the site and size of the tissue tobe resected in a subject. In some embodiments, medical imagingmodalities such as computed tomography (CT) and magnetic resonanceimaging (MM) are used in combination with the methods described herein.In some embodiments, a position emission tomography (PET) is used incombination with the methods described herein. For example, an initialscanning of a cancer patient and/or imaging of a tissue sample from acancer patient can be performed using, e.g., MM, CT, and/or PET prior tothe methods described herein, and a preliminary assessment of a surgicalmargin can be performed. The initial information can provide guidanceon, e.g., where to obtain the tissue sample for use in the methodsdescribed herein, the size of the tissue sample, and/or the number oftissue samples needed. In another example, a follow-up scanning and/orimaging can be performed using e.g., MM, CT, and/or PET after themethods described herein are performed. The follow-up scanning and/orimaging provide information on, e.g., the clearance of the cancerousand/or diseased tissue, and whether there are residual cancerous and/ordiseased tissue. Any other suitable methods known in the art can also beused in combination with the methods described herein.

Some embodiments of any of the methods described herein can furtherinclude obtaining the tissue sample from the subject (e.g., obtain abiopsy sample from the subject).

In some embodiments of any of the methods described herein, at least aportion of the tissue to be resected includes cancer cell(s),pre-cancerous cell(s), necrotic cell(s), infected cell(s), and/ordiseased tissue. In some embodiments, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, or 100% of the tissue to beresected includes one or more of cancer cell(s), pre-cancerous cell(s),necrotic cell(s), infected cell(s), and disease tissue.

In some embodiments of any of the methods described herein, the surgicalmargin can be the margin between the location(s) of one or more ofcancer cell(s), pre-cancerous cell(s), necrotic cell(s), infectedcell(s), and disease tissue, and the location(s) of healthy or normaltissue, in a subject.

In some embodiments, the methods described herein comprise excising anerve and/or blood vessel that is within the determined surgical marginin the subject. In some embodiments, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% of the nerve and/or bloodvessel that is within the determined surgical margin are excised.

In some embodiments, the excision of a nerve and/or blood vessel isconsidered successful when less than 20%, less than 15%, less than 10%,less than 5%, or zero nerve and/or blood vessel that is within thedetermined surgical margin is detected post-excision as compared to theidentified nerve and/or blood vessel that is within the determinedsurgical margin prior to the resection.

In some embodiments, the treatment is considered successful when lessthan 20%, less than 15%, less than 10%, less than 5%, or zero nerveand/or blood vessel that is within the determined surgical margin isdetected post-excision as compared to the identified nerve and/or bloodvessel that is within the determined surgical margin prior to theresection.

In some embodiments, the methods described herein comprise avoidingexcision of a nerve and/or blood vessel outside of the determinedsurgical margin in the subject. In some embodiments, no more than 20%,no more than 15%, no more than 10%, no more than 8%, no more than 6%, nomore than 4%, no more than 2%, or no more than 1% of the nerve and/orblood vessel outside of the determined surgical margin is excised.

The term “presence” as used herein refers to the existence and/orlevel(s) of any object(s) (e.g., an analyte) being measured, quantified,and/or compared in any of the methods described herein.

EXEMPLARY EMBODIMENTS

Embodiment 1. A method of determining size and site of a tissue to beresected from a subject, the method comprising:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises (i) a capture domain that bindsspecifically to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at a different location in the tissuesample, and determining the size and site of the tissue to be resectedfrom the subject based on the comparison.

Embodiment 2. The method of Embodiment 1, wherein the tissue to beresected is a tumor.

Embodiment 3. The method of Embodiment 1, wherein the tissue to beresected is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 4. The method of Embodiment 1 or 2, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 5. The method of Embodiment 4, wherein the cancer is breastcancer.

Embodiment 6. The method of Embodiment 1, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 7. The method of any one of Embodiments 1-6, wherein theanalyte is RNA.

Embodiment 8. The method of Embodiment 7, wherein the RNA is mRNA.

Embodiment 9. The method of any one of Embodiments 1-6, wherein theanalyte is DNA.

Embodiment 10. The method of Embodiment 9, wherein the DNA is genomicDNA.

Embodiment 11. The method of any one of Embodiments 1-10, wherein thearray comprises a slide having the plurality of capture probes.

Embodiment 12. The method of any one of Embodiments 1-10, wherein thearray is a bead array.

Embodiment 13. The method of any one of Embodiments 1-12, wherein step(b) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.

Embodiment 14. The method of Embodiment 13, wherein the sequencing ishigh throughput sequencing.

Embodiment 15. The method of any one of Embodiments 1-12, wherein step(b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 16. The method of Embodiment 15, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising a nucleicacid sequence that is complementary to all or a part of the extendedcapture probe.

Embodiment 17. A method of determining size and site of a tissue to beresected from a subject, the method comprising:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing the presence of the analyte at the location in the tissuesample to the presence of the analyte at different location(s) in thetissue sample, and determining the size and site of the tissue to beresected from the subject based on the comparison.

Embodiment 18. The method of Embodiment 17, wherein the tissue to beresected is a tumor.

Embodiment 19. The method of Embodiment 17, wherein the tissue to beresected is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 20. The method of Embodiment 17 or 18, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 21. The method of Embodiment 20, wherein the cancer is breastcancer.

Embodiment 22. The method of Embodiment 17, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 23. The method of any one of Embodiments 17-22, wherein theanalyte is a protein.

Embodiment 24. The method of Embodiment 23, wherein the analyte is anintracellular protein.

Embodiment 25. The method of Embodiment 23, wherein the analyte is anextracellular protein.

Embodiment 26. The method of any one of Embodiments 23-25, wherein theanalyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 27. The method of any one of Embodiments 17-26, wherein thearray comprises a slide having the plurality of capture probes.

Embodiment 28. The method of any one of Embodiments 17-26, wherein thearray is a bead array.

Embodiment 29. The method of any one of Embodiments 17-28, wherein step(c) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof.

Embodiment 30. The method of Embodiment 29, wherein the sequencing ishigh throughput sequencing.

Embodiment 31. The method of any one of Embodiments 17-30, wherein step(c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 32. The method of Embodiment 31, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 33. A method of treating a subject in need thereof, themethod comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises (i) a capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at a different location in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 34. The method of Embodiment 33, wherein the resected tissueis a tumor.

Embodiment 35. The method of Embodiment 33, wherein the resected tissueis infected tissue, necrotic tissue, or diseased tissue.

Embodiment 36. The method of Embodiment 33 or 34, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 37. The method of Embodiment 36, wherein the cancer is breastcancer.

Embodiment 38. The method of Embodiment 33, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 39. The method of any one of Embodiments 33-38, wherein theanalyte is RNA.

Embodiment 40. The method of Embodiment 39, wherein the RNA is mRNA.

Embodiment 41. The method of any one of Embodiments 33-38, wherein theanalyte is DNA.

Embodiment 42. The method of Embodiment 41, wherein the DNA is genomicDNA.

Embodiment 43. The method of any one of Embodiments 33-42, wherein thearray comprises a slide having the plurality of capture probes.

Embodiment 44. The method of any one of Embodiments 33-42, wherein thearray is a bead array.

Embodiment 45. The method of any one of Embodiments 33-44, wherein step(b) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.

Embodiment 46. The method of Embodiment 45, wherein the sequencing ishigh throughput sequencing.

Embodiment 47. The method of any one of Embodiments 33-46, wherein step(b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 48. The method of Embodiment 47, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 49. A method of treating a subject in need thereof, themethod comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 50. The method of Embodiment 49, wherein the resected tissueis a tumor.

Embodiment 51. The method of Embodiment 49, wherein the resected tissueis infected tissue, necrotic tissue, or diseased tissue.

Embodiment 52. The method of Embodiment 49 or 50, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 53. The method of Embodiment 52, wherein the cancer is breastcancer.

Embodiment 54. The method of Embodiment 49, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 55. The method of any one of Embodiments 49-54, wherein theanalyte is a protein.

Embodiment 56. The method of Embodiment 55, wherein the protein is anintracellular protein.

Embodiment 57. The method of Embodiment 55, wherein the protein is anextracellular protein.

Embodiment 58. The method of any one of Embodiments 55-57, wherein theanalyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 59. The method of any one of Embodiments 49-58, wherein thearray comprises a slide having the plurality of capture probes.

Embodiment 60. The method of any one of Embodiments 49-58, wherein thearray is a bead array.

Embodiment 61. The method of any one of Embodiments 49-60, wherein step(c) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof. Embodiment62. The method of Embodiment 61, wherein the sequencing is highthroughput sequencing.

Embodiment 63. The method of any one of Embodiments 49-62, wherein step(c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 64. The method of Embodiment 63, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 65. A method of identifying a surgical margin of a tissue tobe resected in a subject, the method comprising:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises (i) a capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin of the tissue to be resectedfrom the subject based on the comparison.

Embodiment 66. The method of Embodiment 65, wherein the tissue to beresected is a tumor.

Embodiment 67. The method of Embodiment 65, wherein the tissue to beresected is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 68. The method of Embodiment 65 or 66, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 69. The method of Embodiment 68, wherein the cancer is breastcancer.

Embodiment 70. The method of Embodiment 65, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 71. The method of any one of Embodiments 65-70, wherein theanalyte is RNA.

Embodiment 72. The method of Embodiment 71, wherein the RNA is mRNA.

Embodiment 73. The method of any one of Embodiments 65-70, wherein theanalyte is DNA.

Embodiment 74. The method of Embodiment 73, wherein the DNA is genomicDNA.

Embodiment 75. The method of any one of Embodiments 65-74, wherein thearray comprises a slide.

Embodiment 76. The method of any one of Embodiments 65-74, wherein thearray is a bead array.

Embodiment 77. The method of any one of Embodiments 65-76, wherein step(b) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of the nucleic acidsequence corresponding to the spatial barcode or a complement thereof.

Embodiment 78. The method of Embodiment 77, wherein the sequencing ishigh throughput sequencing.

Embodiment 79. The method of any one of Embodiments 65-78, wherein step(b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 80. The method of Embodiment 79, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 81. A method of identifying a surgical margin of a tissue tobe resected in a subject, the method comprising:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin of the tissue to be resectedfrom the subject based on the comparison.

Embodiment 82. The method of Embodiment 81, wherein the tissue to beresected is a tumor.

Embodiment 83. The method of Embodiment 81, wherein the tissue to beresected is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 84. The method of Embodiment 81 or 82, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 85. The method of Embodiment 84, wherein the cancer is breastcancer.

Embodiment 86. The method of Embodiment 81, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 87. The method of any one of Embodiments 81-86, wherein theanalyte is a protein.

Embodiment 88. The method of Embodiment 87, wherein the protein is anintracellular protein.

Embodiment 89. The method of Embodiment 87, wherein the protein is anextracellular protein.

Embodiment 90. The method of any one of Embodiments 87-89, wherein theanalyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 91. The method of any one of Embodiments 81-90, wherein thearray comprises a slide.

Embodiment 92. The method of any one of Embodiments 81-90, wherein thearray is a bead array.

Embodiment 93. The method of any one of Embodiments 81-92, wherein step(c) comprises sequencing (i) all or a part of the nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof.

Embodiment 94. The method of Embodiment 93, wherein the sequencing ishigh throughput sequencing.

Embodiment 95. The method of any one of Embodiments 81-94, wherein step(c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 96. The method of Embodiment 95, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 97. A method of reducing the risk of re-excision of a tissuefrom a subject, the method comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 98. The method of Embodiment 97, wherein the resected tissueis a tumor.

Embodiment 99. The method of Embodiment 97, wherein the resected tissueis infected tissue, necrotic tissue, or diseased tissue.

Embodiment 100. The method of Embodiment 97 or 98, wherein the subjectis suspected of or diagnosed as having a cancer.

Embodiment 101. The method of Embodiment 100, wherein the cancer isbreast cancer.

Embodiment 102. The method of Embodiment 97, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 103. The method of any one of Embodiments 97-102, wherein theanalyte is RNA.

Embodiment 104. The method of Embodiment 103, wherein the RNA is mRNA.

Embodiment 105. The method of any one of Embodiments 97-102, wherein theanalyte is DNA.

Embodiment 106. The method of Embodiment 105, wherein the DNA is genomicDNA.

Embodiment 107. The method of any one of Embodiments 97-106, wherein thearray comprises a slide having the plurality of capture probes.

Embodiment 108. The method of any one of Embodiments 97-106, wherein thearray is a bead array.

Embodiment 109. The method of any one of Embodiments 97-108, whereinstep (b) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte specifically bound to the capturedomain or a complement thereof, and (ii) all or a part of the nucleicacid sequence corresponding to the spatial barcode or a complementthereof.

Embodiment 110. The method of Embodiment 109, wherein the sequencing ishigh throughput sequencing.

Embodiment 111. The method of any one of Embodiments 97-110, whereinstep (b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 112. The method of Embodiment 111, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 113. A method of reducing the risk of re-excision of a tissuefrom a subject, the method comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 114. The method of Embodiment 113, wherein the resectedtissue is a tumor.

Embodiment 115. The method of Embodiment 113, wherein the resectedtissue is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 116. The method of Embodiment 113 or 114, wherein the subjectis suspected of or diagnosed as having a cancer.

Embodiment 117. The method of Embodiment 116, wherein the cancer isbreast cancer.

Embodiment 118. The method of Embodiment 113, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 119. The method of any one of Embodiments 113-118, whereinthe analyte is a protein.

Embodiment 120. The method of Embodiment 119, wherein the protein isintracellular.

Embodiment 121. The method of Embodiment 119, wherein the protein isextracellular.

Embodiment 122. The method of any one of Embodiments 119-121, whereinthe analyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 123. The method of any one of Embodiments 113-122, whereinthe array comprises a slide having the plurality of capture probes.

Embodiment 124. The method of any one of Embodiments 113-122, whereinthe array is a bead array.

Embodiment 125. The method of any one of Embodiments 113-124, whereinstep (c) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof.

Embodiment 126. The method of Embodiment 125, wherein the sequencing ishigh throughput sequencing.

Embodiment 127. The method of any one of Embodiments 113-126, whereinstep (c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 128. The method of Embodiment 127, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 129. A method of reducing the rate of recurrence of a tissueabnormality in a subject, the method comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 130. The method of Embodiment 129, wherein the resectedtissue is a tumor.

Embodiment 131. The method of Embodiment 129, wherein the resectedtissue is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 132. The method of Embodiment 129 or 130, wherein the subjectis suspected of or diagnosed as having a cancer.

Embodiment 133. The method of Embodiment 132, wherein the cancer isbreast cancer.

Embodiment 134. The method of Embodiment 129, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 135. The method of any one of Embodiments 129-134, whereinthe analyte is RNA.

Embodiment 136. The method of Embodiment 135, wherein the RNA is mRNA.

Embodiment 137. The method of any one of Embodiments 129-134, whereinthe analyte is DNA.

Embodiment 138. The method of Embodiment 137, wherein the DNA is genomicDNA.

Embodiment 139. The method of any one of Embodiments 129-138, whereinthe array comprises a slide having the plurality of capture probes.

Embodiment 140. The method of any one of Embodiments 129-138, whereinthe array is a bead array.

Embodiment 141. The method of any one of Embodiments 129-140, whereinstep (b) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte specifically bound to the capturedomain or a complement thereof, and (ii) all or a part of the nucleicacid sequence corresponding to the spatial barcode or a complementthereof.

Embodiment 142. The method of Embodiment 141, wherein the sequencing ishigh throughput sequencing.

Embodiment 143. The method of any one of Embodiments 129-142, whereinstep (b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 144. The method of Embodiment 143, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 145. A method of reducing the rate of recurrence of a tissueabnormality in a subject, the method comprising:

resecting tissue from the subject using a surgical margin previouslydetermined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 146. The method of Embodiment 145, wherein the resectedtissue is a tumor.

Embodiment 147. The method of Embodiment 145, wherein the resectedtissue is infected tissue, necrotic tissue, or diseased tissue.

Embodiment 148. The method of Embodiment 145 or 146, wherein the subjectis suspected of or diagnosed as having a cancer.

Embodiment 149. The method of Embodiment 148, wherein the cancer isbreast cancer.

Embodiment 150. The method of Embodiment 145, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 151. The method of any one of Embodiments 145-150, whereinthe analyte is a protein.

Embodiment 152. The method of Embodiment 151, wherein the protein is anintracellular protein.

Embodiment 153. The method of Embodiment 151, wherein the protein is anextracellular protein.

Embodiment 154. The method of any one of Embodiments 151-153, whereinthe analyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 155. The method of any one of Embodiments 145-154, whereinthe array comprises a slide having the plurality of capture probes.

Embodiment 156. The method of any one of Embodiments 145-154, whereinthe array is a bead array.

Embodiment 157. The method of any one of Embodiments 145-156, whereinstep (c) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof.

Embodiment 158. The method of Embodiment 157, wherein the sequencing ishigh throughput sequencing.

Embodiment 159. The method of any one of Embodiments 145-158, whereinstep (c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 160. The method of Embodiment 159, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 161. A method of treating a subject, the method comprising:

excising a nerve or blood vessel that is within a surgical margin in thesubject, or avoiding excision of a nerve or blood vessel outside of thesurgical margin in the subject, wherein the surgical margin waspreviously determined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to an arraycomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a (i) capture domain thatspecifically binds to an analyte of the tissue sample and (ii) a spatialbarcode;

(b) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte specifically bound to the capture domain ora complement thereof, and (ii) all or a part of a nucleic acid sequencecorresponding to the spatial barcode or a complement thereof, and usingthe determined nucleic acid sequences of (i) and (ii) to identify thepresence of the analyte at a location in the tissue sample;

(c) comparing the presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 162. The method of Embodiment 161, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 163. The method of Embodiment 162, wherein the cancer isbreast cancer.

Embodiment 164. The method of Embodiment 161, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 165. The method of any one of Embodiments 161-164, whereinthe analyte is RNA.

Embodiment 166. The method of Embodiment 165, wherein the RNA is mRNA.

Embodiment 167. The method of any one of Embodiments 161-164, whereinthe analyte is DNA.

Embodiment 168. The method of Embodiment 167, wherein the DNA is genomicDNA.

Embodiment 169. The method of any one of Embodiments 161-168, whereinthe array comprises a slide having the plurality of capture probes.

Embodiment 170. The method of any one of Embodiments 161-169, whereinthe array is a bead array.

Embodiment 171. The method of any one of Embodiments 161-170, whereinstep (b) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte specifically bound to the capturedomain or a complement thereof, and (ii) all or a part of the nucleicacid sequence corresponding to the spatial barcode or a complementthereof.

Embodiment 172. The method of Embodiment 171, wherein the sequencing ishigh throughput sequencing.

Embodiment 173. The method of any one of Embodiments 161-172, whereinstep (b) comprises extending a 3′ end of the capture probe using thespecifically bound analyte as a template to generate an extended captureprobe.

Embodiment 174. The method of Embodiment 173, wherein step (b) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 175. The method of any one of Embodiments 161-174, whereinthe method excising a nerve or blood vessel that is within the surgicalmargin in the subject.

Embodiment 176. The method of any one of Embodiments 161-174, whereinthe method comprises avoiding excision of a nerve or blood vesseloutside of the surgical margin in the subject.

Embodiment 177. A method of treating a subject, the method comprising:

excising a nerve or blood vessel that is within a surgical margin in thesubject, or avoiding excision of a nerve or blood vessel outside of thesurgical margin in the subject, wherein the surgical margin waspreviously determined using a method comprising the steps of:

(a) contacting a tissue sample obtained from the subject to a pluralityof analyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises an analyte binding moietybarcode, an analyte capture sequence, and an analyte binding moiety thatbinds specifically to an analyte;

(b) disposing the tissue sample onto an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain that binds specifically to the analyte capture sequence;

(c) determining (i) all or a part of a nucleic acid sequencecorresponding to the analyte binding moiety barcode or a complementthereof, and (ii) all or a part of a nucleic acid sequence correspondingto the spatial barcode or a complement thereof, and using the determinednucleic acid sequences of (i) and (ii) to identify the presence of theanalyte at a location in the tissue sample;

(d) comparing presence of the analyte at the location in the tissuesample to presence of the analyte at different location(s) in the tissuesample, and determining the surgical margin based on the comparison.

Embodiment 178. The method of Embodiment 177, wherein the subject issuspected of or diagnosed as having a cancer.

Embodiment 179. The method of Embodiment 178, wherein the cancer isbreast cancer.

Embodiment 180. The method of Embodiment 177, wherein the subject issuspected of or diagnosed as having ductal carcinoma in situ.

Embodiment 181. The method of any one of Embodiments 177-180, whereinthe analyte is a protein.

Embodiment 182. The method of Embodiment 181, wherein the protein is anintracellular protein.

Embodiment 183. The method of Embodiment 181, wherein the protein is anextracellular protein.

Embodiment 184. The method of any one of Embodiments 181-183, whereinthe analyte binding moiety is an antibody or an antigen-binding antibodyfragment.

Embodiment 185. The method of any one of Embodiments 177-184, whereinthe array comprises a slide having the plurality of capture probes.

Embodiment 186. The method of any one of Embodiments 177-184, whereinthe array is a bead array.

Embodiment 187. The method of any one of Embodiments 177-186, whereinstep (c) comprises sequencing (i) all or a part of the nucleic acidsequence corresponding to the analyte binding moiety barcode or acomplement thereof, and (ii) all or a part of the nucleic acid sequencecorresponding to the spatial barcode or a complement thereof.

Embodiment 188. The method of Embodiment 187, wherein the sequencing ishigh throughput sequencing.

Embodiment 189. The method of any one of Embodiments 177-188, whereinstep (c) comprises extending a 3′ end of the capture probe using thespecifically bound analyte capture agent as a template to generate anextended capture probe.

Embodiment 190. The method of Embodiment 189, wherein step (c) furthercomprises generating a single-stranded nucleic acid comprising asequence that is complementary to all or a part of the extended captureprobe.

Embodiment 191. The method of any one of Embodiments 177-190, whereinthe method comprises excising a nerve or blood vessel that is within thesurgical margin in the subject.

Embodiment 192. The method of any one of Embodiments 177-190, whereinthe method comprises avoiding excision of a nerve or blood vesseloutside of the surgical margin in the subject.

What is claimed is:
 1. A method of determining a surgical margin of atissue to be resected in a subject, the method comprising: (a)contacting a tissue sample obtained from the subject to a plurality ofanalyte capture agents, wherein an analyte capture agent of theplurality of analyte capture agents comprises: an analyte binding moietythat binds specifically to an analyte, an analyte binding moietybarcode, and an analyte capture sequence; (b) contacting the tissuesection obtained from the subject to a substrate comprising a pluralityof capture probes affixed to the substrate, wherein a capture probe ofthe plurality of capture probes comprises (i) a capture domain thathybridizes to the analyte capture sequence and (ii) a spatial barcode;(c) permeabilizing the tissue section and hybridizing the analytecapture sequence to the capture domain; (d) determining (i) all or apart of a nucleic acid sequence corresponding to the analyte hybridizedto the capture domain or a complement thereof, and (ii) the spatialbarcode or a complement thereof, and using the determined nucleic acidsequences of (i) and (ii) to identify the presence of the analyte at alocation in the tissue section; and (e) comparing the presence of theanalyte at the location in the tissue section to presence of the analyteat one or more different locations in the tissue section, anddetermining the surgical margin of the tissue to be resected from thesubject based on the comparison.
 2. The method of claim 1, wherein thepresence of the analyte at the location in the tissue section isincreased compared to the presence of the analyte at the differentlocation in the tissue section.
 3. The method of claim 1, wherein thepresence of the analyte at the location in the tissue section isdecreased compared to the presence of the analyte at the differentlocation in the tissue section.
 4. The method of claim 1, wherein thedifferent locations in the tissue section are reference locations. 5.The method of claim 4, wherein the reference locations in the tissuesection are locations of non-cancerous tissue and/or non-tumor tissue.6. The method of claim 1, wherein the tissue to be resected is a tumor.7. The method of claim 1, wherein the tissue to be resected is aninfected tissue, a necrotic tissue, or a diseased tissue.
 8. The methodof claim 7, wherein the tissue to be resected is infected by abacterium, a virus, a fungus, a parasite, and/or protozoa.
 9. The methodof claim 1, wherein the subject is suspected of or diagnosed as having acancer.
 10. The method of claim 9, wherein the cancer is breast cancer.11. The method of claim 1, wherein the subject is suspected of ordiagnosed as having ductal carcinoma in situ.
 12. The method of claim 1,wherein the analyte is a protein.
 13. The method of claim 1, wherein theanalyte is a tumor biomarker and/or a tumor antigen.
 14. The method ofclaim 1, wherein the array is a bead array.
 15. The method of claim 1,wherein step (c) comprises sequencing (i) all or a part of the nucleicacid sequence corresponding to the analyte specifically bound to thecapture domain or a complement thereof, and (ii) the spatial barcode ora complement thereof.
 16. The method of claim 15, wherein the sequencingcomprises high throughput sequencing.
 17. The method of claim 1, whereinstep (c) comprises extending a 3′ end of the capture probe using theanalyte as a template to generate an extended capture probe.
 18. Themethod of claim 17, wherein step (c) further comprises generating asingle-stranded nucleic acid comprising a nucleic acid sequence that iscomplementary to all or a part of the extended capture probe.
 19. Themethod of claim 1, wherein the capture probe further comprises one ormore functional domains, a unique molecular identifier, a cleavagedomain, or combinations thereof.
 20. The method of claim 1, wherein thecapture domain comprises a poly-uridine sequence or a poly-thymidinesequence.
 21. The method of claim 1, wherein comparing the presence ofthe analyte at the location in the tissue section to the presence of theanalyte at one or more different locations in the tissue section furthercomprises determining the size and site of the tissue to be resectedfrom the subject.
 22. The method of claim 1, wherein the method resultsin a reduced rate of re-excision of the tissue.
 23. The method of claim1, wherein the method results in a reduced rate of recurrence of thetissue abnormality in the subject.
 24. The method of claim 1, whereinthe method further comprises treatment of the subject by surgery afterstep (d).
 25. The method of claim 1, wherein the tissue section wasobtained from the subject.
 26. The method of claim 1, wherein the tissuesection was previously fixed and/or stained.
 27. The method of claim 26,wherein the tissue section was previously stained by hematoxylin andeosin, immunohistochemistry, and/or immunofluorescence.
 28. The methodof claim 1, further comprising identifying the location of a secondanalyte in the tissue sample, the method further comprising: hybridizingthe second analyte to the capture domain; and determining (iii) all or apart of a second analyte hybridized to the capture domain or acomplement thereof, and (iv) the spatial barcode or a complementthereof, and using the determined nucleic acid sequences of (iii) and(iv) to identify the location of the second analyte in the tissuesample.
 29. The method of claim 28, further comprising extending a 3′end of the capture probe using the second analyte as a template togenerate a second extended capture probe.
 30. The method of claim 28,wherein the second analyte is RNA.